*Corresponding author: Hossein Hamzeiy (PhD) and Jaleh Barar (PhD), Tel.: + 98 411 3367914, Fax: +98 411 3367929, E-mail: [email protected], [email protected] Copyright © 2011 by Tabriz University of Medical Sciences
BioImpacts, 2011, 1(2), 113-119 doi: 10.5681/bi.2011.015 http://bi.tbzmed.ac.ir/
Gap Junctions: The Claymore for Cancerous Cells Masoud Asadi-Khiavi1,2,3, Hossein Hamzeiy1,2*, Sajjad Khani2, Ailar Nakhlband2 and Jaleh Barar2,4* 1Department of Pharmacology and Toxicology, School of Pharmacy, Tabriz University of Medical Sciences, Tabriz, Iran 2Research Center for Pharmaceutical Nanotechnology, School of Pharmacy, Tabriz University of Medical Sciences, Tabriz, Iran 3Student Research Committee, Tabriz University of Medical Sciences, Tabriz, Iran 4Ovarian Cancer Research Center, School of Medicine, University of Pennsylvania, Philadelphia, USA
Introduction
Cancer is a terrible disorder facing human today and unfortunately, lack of deep knowledge about cancer biology causes perfect treatment failing. However, several strategies have been developed upon recent findings in molecular biology. One of these approaches appears to be valuable because of inhibiting inter-cellular gap junction communication (GJIC) between cells. As a rule, lack of gap junctions was commonly observed in carcinogenesis (Leithe et al. 2006). Some critical small biomolecules like cAMP and Ca2+ as well as several small sized amino acids could pass through this communicative channel. Cellular apoptosis pathway, Akt signaling pathway, EGF pathway, p53 pathway, inhibition of matrix metalloproteinases (MMPs) and VEGF family ligands and receptors are the most important biomolecules and ways in cancer biology which could be affected by these small agents. As
mentioned, EGF pathway and its obedience biomolecules has vital role in carcinogenesis of epithelial derived carcinomas like breast and lung cancers (Cameron et al. 2003). This cell-surface tyrosine kinase receptor is a member of the ErbB family of receptors and mutations which could result in some epithelial derived carcinomas including breast cancer through affecting its expression or activity. EGFR over expression was reported in 20% - 50% of breast cancer (Agelaki et al. 2009). EGFR is activated by binding its specific ligands, including epidermal growth factor (EGF) and transforming growth factor α (TGFα). Due to activation by its ligands, EGFR switches from an inactive monomeric form to an active homodimer (Dong J and Wiley HS 2000). Several signal transduction cascades was initiated by downstream signaling proteins, principally the mitogen-activated protein kinase (MAPK), phosphatidylinositol-3-kinase (PI-3K/Akt), and finally signal transducer and activator of
A B S T R A C T A R T I C L E I N F O
Keywords: Cancer Cells EGFR Gap Junction Cytotoxicity Gene Expression
Article History: Received: 14 July 2011 Revised: 17 July 2011 Accepted: 20 July 2011 ePublished: 31 July 2011
Article Type: Research Article
Introduction: Gap junctions play an important role in the cell proliferation in mammalian cells as well as carcinogenesis. However, there are controversial issues about their role in cancer pathogenesis. This study was designed to evaluate genotoxicity and cytotoxicity of Carbenoxolone (CBX) as a prototype of inter-cellular gap junction blocker in MCF7 and BT20 human breast cancer cells. Methods: The MCF7and BT20 human breast cancer cell lines were cultivated, and treated at designated confluency with different doses of CBX. Cellular cytotoxicity was examined using standard colorimetric assay associated with cell viability tests. Gene expression evaluation was carried out using real time polymerase chain reaction (PCR). Results: MCF7 and BT20 cells were significantly affected by CBX in a dose dependent manner in cell viability assays. Despite varying expression of genes, down regulation of pro- and anti-apoptotic genes was observed in these cells. Conclusion: Based upon this investigation, it can be concluded that CBX could affect both low and high proliferative types of breast cancer cell lines and disproportionate down regulation of both pre- and anti-apoptotic genes may be related to interacting biomolecules, perhaps via gap junctions.
junctions
Asadi
114
transcription (STAT) pathways et al2006)adhesion and migrationinvolvement in the metastasis, angiogenesis and apoptosis inhibition 2005)it is believed that EGFR signal transduction could be modulatetransferredpromote (C34H50O7) [3Oleancellularjunctionsseveral derivatiand demonstrates a superficial resemblance to steroid structuresinhibitor 2001about Crespin determine the impacts of CBX on MCF7and BT20 human breast cancer cells in the view of apoptosis
Materials and
MaterialsThe MCF7Cell culture plates and flasks were obtained from National cell bank of Iran (Pasteur institute, Iran) and IWAKI, Japan respectively. RPMI1640 medium, TrypsinDithiotheritol (DTT), first strand reverse transcriptase (RT) buffer, Moloney murine leukemia virus reversetranscriptase (MMLVSigma Aldrich Co., (Poole, UK). Agarose and Fetal bovine serum (FBS) were bought from Gibco, Invitrogen (Paisley, UK). Ribonuclease inhibitor was obtained fromFermentas, Canada. (Glyceraldehyde 3
Asadi-Khiavi
114 | BioImpacts
transcription (STAT) pathways et al. 2008; Camp 2006) adhesion and migrationinvolvement in the metastasis, angiogenesis and apoptosis inhibition 2005). it is believed that EGFR signal transduction could be modulatetransferredpromote (C34H50O7) [3Olean-12 encellularjunctionsseveral derivatiand demonstrates a superficial resemblance to steroid structuresinhibitor 2001). about Crespin determine the impacts of CBX on MCF7and BT20 human breast cancer cells in the view of apoptosis
Materials and
MaterialsThe MCF7Cell culture plates and flasks were obtained from National cell bank of Iran (Pasteur institute, Iran) and IWAKI, Japan respectively. RPMI1640 medium, TrypsinDithiotheritol (DTT), first strand reverse transcriptase (RT) buffer, Moloney murine leukemia virus reversetranscriptase (MMLVSigma Aldrich Co., (Poole, UK). Agarose and Fetal bovine serum (FBS) were bought from Gibco, Invitrogen (Paisley, UK). Ribonuclease inhibitor was obtained fromFermentas, Canada. (Glyceraldehyde 3
Khiavi
BioImpacts
transcription (STAT) pathways . 2008; Camp which mainly undergoes to cell proliferation,
adhesion and migrationinvolvement in the metastasis, angiogenesis and apoptosis inhibition
. In regards to these miscellaneous biomoleculesit is believed that EGFR signal transduction could be modulatedtransferredpromote pathogenesis(C34H50O7) [3
12 encellular communicationjunctions, inhibits cell growthseveral types of derivation and demonstrates a superficial resemblance to steroid structures. inhibitor in cells
. However, there are some controversialabout gap junction Crespin et aldetermine the impacts of CBX on MCF7and BT20 human breast cancer cells in the view of apoptosis particularly
Fig.
Materials and
Materials The MCF7Cell culture plates and flasks were obtained from National cell bank of Iran (Pasteur institute, Iran) and IWAKI, Japan respectively. RPMI1640 medium, Trypsin-EDTA (0.05%Dithiotheritol (DTT), first strand reverse transcriptase (RT) buffer, Moloney murine leukemia virus reversetranscriptase (MMLVSigma Aldrich Co., (Poole, UK). Agarose and Fetal bovine serum (FBS) were bought from Gibco, Invitrogen (Paisley, UK). Ribonuclease inhibitor was obtained fromFermentas, Canada. (Glyceraldehyde 3
Khiavi et al
BioImpacts
transcription (STAT) pathways . 2008; Camp
which mainly undergoes to cell proliferation, adhesion and migrationinvolvement in the metastasis, angiogenesis and apoptosis inhibition
n regards to these miscellaneous biomoleculesit is believed that EGFR signal transduction could be
d by transferred via gap junction
pathogenesis(C34H50O7) [3
12 en-29communication, inhibits cell growthtypes of of
and demonstrates a superficial resemblance to steroid This drug
in cellsHowever, there are some controversialgap junction
et aldetermine the impacts of CBX on MCF7and BT20 human breast cancer cells in the view of
particularly
Fig. 1.
Materials and
The MCF7 and BT20 human breast cancer cell lines, Cell culture plates and flasks were obtained from National cell bank of Iran (Pasteur institute, Iran) and IWAKI, Japan respectively. RPMI1640 medium,
EDTA (0.05%Dithiotheritol (DTT), first strand reverse transcriptase (RT) buffer, Moloney murine leukemia virus reversetranscriptase (MMLVSigma Aldrich Co., (Poole, UK). Agarose and Fetal bovine serum (FBS) were bought from Gibco, Invitrogen (Paisley, UK). Ribonuclease inhibitor was obtained fromFermentas, Canada. (Glyceraldehyde 3
et al.
BioImpacts, 2011, 1(2),
transcription (STAT) pathways . 2008; Camp
which mainly undergoes to cell proliferation, adhesion and migrationinvolvement in the metastasis, angiogenesis and apoptosis inhibition
n regards to these miscellaneous biomoleculesit is believed that EGFR signal transduction could be
by potentvia gap junction
pathogenesis(C34H50O7) [3
29-oic acid] (Fig. 1) as communication, inhibits cell growthtypes of
of Glycyrrhizin and demonstrates a superficial resemblance to steroid
This drug in cells
However, there are some controversialgap junction
et al. 2009)determine the impacts of CBX on MCF7and BT20 human breast cancer cells in the view of
particularly
1. The structure of carbenoxolone (CBX)
Materials and methods
and BT20 human breast cancer cell lines, Cell culture plates and flasks were obtained from National cell bank of Iran (Pasteur institute, Iran) and IWAKI, Japan respectively. RPMI1640 medium,
EDTA (0.05%Dithiotheritol (DTT), first strand reverse transcriptase (RT) buffer, Moloney murine leukemia virus reversetranscriptase (MMLVSigma Aldrich Co., (Poole, UK). Agarose and Fetal bovine serum (FBS) were bought from Gibco, Invitrogen (Paisley, UK). Ribonuclease inhibitor was obtained fromFermentas, Canada. (Glyceraldehyde 3
, 2011, 1(2),
transcription (STAT) pathways . 2008; Camp et al
which mainly undergoes to cell proliferation, adhesion and migrationinvolvement in the metastasis, angiogenesis and apoptosis inhibition
n regards to these miscellaneous biomoleculesit is believed that EGFR signal transduction could be
potentvia gap junction
pathogenesis(C34H50O7) [3-
oic acid] (Fig. 1) as communication, inhibits cell growthtypes of cancer
Glycyrrhizin and demonstrates a superficial resemblance to steroid
This drug (Ye, Z. C.,
However, there are some controversialgap junction
. 2009)determine the impacts of CBX on MCF7and BT20 human breast cancer cells in the view of
particularly
The structure of carbenoxolone (CBX)
methods
and BT20 human breast cancer cell lines, Cell culture plates and flasks were obtained from National cell bank of Iran (Pasteur institute, Iran) and IWAKI, Japan respectively. RPMI1640 medium,
EDTA (0.05%Dithiotheritol (DTT), first strand reverse transcriptase (RT) buffer, Moloney murine leukemia virus reversetranscriptase (MMLVSigma Aldrich Co., (Poole, UK). Agarose and Fetal bovine serum (FBS) were bought from Gibco, Invitrogen (Paisley, UK). Ribonuclease inhibitor was obtained fromFermentas, Canada. (Glyceraldehyde 3-phosphate dehydrogenase)
, 2011, 1(2),
transcription (STAT) pathways et al
which mainly undergoes to cell proliferation, adhesion and migration (involvement in the metastasis, angiogenesis and apoptosis inhibition (Gialeli
n regards to these miscellaneous biomoleculesit is believed that EGFR signal transduction could be
potent small moleculesvia gap junction
pathogenesis of cancer-(3-carboxy
oic acid] (Fig. 1) as communication blocker , inhibits cell growth
cancerGlycyrrhizin
and demonstrates a superficial resemblance to steroid This drug is often used as a gap junction
Ye, Z. C., However, there are some controversialgap junction role in
. 2009). determine the impacts of CBX on MCF7and BT20 human breast cancer cells in the view of
particularly at mRNA
The structure of carbenoxolone (CBX)
methods
and BT20 human breast cancer cell lines, Cell culture plates and flasks were obtained from National cell bank of Iran (Pasteur institute, Iran) and IWAKI, Japan respectively. RPMI1640 medium,
EDTA (0.05%Dithiotheritol (DTT), first strand reverse transcriptase (RT) buffer, Moloney murine leukemia virus reversetranscriptase (MMLV-rt, 200U/Sigma Aldrich Co., (Poole, UK). Agarose and Fetal bovine serum (FBS) were bought from Gibco, Invitrogen (Paisley, UK). Ribonuclease inhibitor was obtained fromFermentas, Canada.
phosphate dehydrogenase)
, 2011, 1(2), 113
transcription (STAT) pathways et al. 2005;
which mainly undergoes to cell proliferation, (Agelaki
involvement in the metastasis, angiogenesis and Gialeli
n regards to these miscellaneous biomoleculesit is believed that EGFR signal transduction could be
small moleculesvia gap junction
of cancercarboxy
oic acid] (Fig. 1) as blocker
, inhibits cell growthcancer.
Glycyrrhizin obtained from Licorice root and demonstrates a superficial resemblance to steroid
is often used as a gap junction Ye, Z. C.,
However, there are some controversialrole in. This study was designed to
determine the impacts of CBX on MCF7and BT20 human breast cancer cells in the view of
at mRNA
The structure of carbenoxolone (CBX)
and BT20 human breast cancer cell lines, Cell culture plates and flasks were obtained from National cell bank of Iran (Pasteur institute, Iran) and IWAKI, Japan respectively. RPMI1640 medium,
EDTA (0.05%Dithiotheritol (DTT), first strand reverse transcriptase (RT) buffer, Moloney murine leukemia virus reverse
rt, 200U/Sigma Aldrich Co., (Poole, UK). Agarose and Fetal bovine serum (FBS) were bought from Gibco, Invitrogen (Paisley, UK). Ribonuclease inhibitor was obtained fromFermentas, Canada. STAT1,
phosphate dehydrogenase)
113-119
transcription (STAT) pathways 2005;
which mainly undergoes to cell proliferation, Agelaki
involvement in the metastasis, angiogenesis and Gialeli et al
n regards to these miscellaneous biomoleculesit is believed that EGFR signal transduction could be
small moleculesvia gap junctions
of cancercarboxy
oic acid] (Fig. 1) as blocker
, inhibits cell growth and CBX is a semiobtained from Licorice root
and demonstrates a superficial resemblance to steroid is often used as a gap junction
Ye, Z. C., et alHowever, there are some controversial
role in This study was designed to
determine the impacts of CBX on MCF7and BT20 human breast cancer cells in the view of
at mRNA
The structure of carbenoxolone (CBX)
and BT20 human breast cancer cell lines, Cell culture plates and flasks were obtained from National cell bank of Iran (Pasteur institute, Iran) and IWAKI, Japan respectively. RPMI1640 medium,
EDTA (0.05%–0.02%), TriDithiotheritol (DTT), first strand reverse transcriptase (RT) buffer, Moloney murine leukemia virus reverse
rt, 200U/Sigma Aldrich Co., (Poole, UK). Agarose and Fetal bovine serum (FBS) were bought from Gibco, Invitrogen (Paisley, UK). Ribonuclease inhibitor was obtained from
STAT1, phosphate dehydrogenase)
119
transcription (STAT) pathways (Loew 2005; Mamot and Rochlitz.
which mainly undergoes to cell proliferation, Agelaki et al
involvement in the metastasis, angiogenesis and et al
n regards to these miscellaneous biomoleculesit is believed that EGFR signal transduction could be
small molecules and could
of cancer (Ncarboxy-1-
oic acid] (Fig. 1) as blocker which mediated
and CBX is a semi
obtained from Licorice root and demonstrates a superficial resemblance to steroid
is often used as a gap junction et al
However, there are some controversial carcinogenesis
This study was designed to determine the impacts of CBX on MCF7and BT20 human breast cancer cells in the view of
at mRNA transcriptomics level.
The structure of carbenoxolone (CBX)
and BT20 human breast cancer cell lines, Cell culture plates and flasks were obtained from National cell bank of Iran (Pasteur institute, Iran) and IWAKI, Japan respectively. RPMI1640 medium,
0.02%), TriDithiotheritol (DTT), first strand reverse transcriptase (RT) buffer, Moloney murine leukemia virus reverse
rt, 200U/l) Sigma Aldrich Co., (Poole, UK). Agarose and Fetal bovine serum (FBS) were bought from Gibco, Invitrogen (Paisley, UK). Ribonuclease inhibitor was obtained from
STAT1, phosphate dehydrogenase)
Loew Mamot and Rochlitz.
which mainly undergoes to cell proliferation, et al. 2009
involvement in the metastasis, angiogenesis and et al. 2009; Camp
n regards to these miscellaneous biomoleculesit is believed that EGFR signal transduction could be
small moleculesand could(Nicholson 2003)
-oxopropoxy)oic acid] (Fig. 1) as
which mediated induces apoptosis
CBX is a semiobtained from Licorice root
and demonstrates a superficial resemblance to steroid is often used as a gap junction
et al. 2003However, there are some controversial
carcinogenesisThis study was designed to
determine the impacts of CBX on MCF7and BT20 human breast cancer cells in the view of
transcriptomics level.
The structure of carbenoxolone (CBX)
and BT20 human breast cancer cell lines, Cell culture plates and flasks were obtained from National cell bank of Iran (Pasteur institute, Iran) and IWAKI, Japan respectively. RPMI1640 medium,
0.02%), TriDithiotheritol (DTT), first strand reverse transcriptase (RT) buffer, Moloney murine leukemia virus reverse
l) wasSigma Aldrich Co., (Poole, UK). Agarose and Fetal bovine serum (FBS) were bought from Gibco, Invitrogen (Paisley, UK). Ribonuclease inhibitor was obtained from
STAT1, AKT1, GAPDH phosphate dehydrogenase)
Loew et alMamot and Rochlitz.
which mainly undergoes to cell proliferation, . 2009
involvement in the metastasis, angiogenesis and . 2009; Camp
n regards to these miscellaneous biomoleculesit is believed that EGFR signal transduction could be
small molecules which may be and could
icholson 2003)oxopropoxy)
oic acid] (Fig. 1) as prototypic which mediated
induces apoptosis CBX is a semi
obtained from Licorice root and demonstrates a superficial resemblance to steroid
is often used as a gap junction 2003
However, there are some controversialcarcinogenesis
This study was designed to determine the impacts of CBX on MCF7and BT20 human breast cancer cells in the view of cell growth and
transcriptomics level.
The structure of carbenoxolone (CBX)
and BT20 human breast cancer cell lines, Cell culture plates and flasks were obtained from National cell bank of Iran (Pasteur institute, Iran) and IWAKI, Japan respectively. RPMI1640 medium,
0.02%), TriDithiotheritol (DTT), first strand reverse transcriptase (RT) buffer, Moloney murine leukemia virus reverse
was purchased from Sigma Aldrich Co., (Poole, UK). Agarose and Fetal bovine serum (FBS) were bought from Gibco, Invitrogen (Paisley, UK). Ribonuclease inhibitor was obtained from
AKT1, GAPDH phosphate dehydrogenase)
et al. 2009; Mamot and Rochlitz.
which mainly undergoes to cell proliferation, . 2009)
involvement in the metastasis, angiogenesis and . 2009; Camp
n regards to these miscellaneous biomoleculesit is believed that EGFR signal transduction could be
which may be and could prevent or
icholson 2003)oxopropoxy)
prototypic which mediated
induces apoptosis CBX is a semi
obtained from Licorice root and demonstrates a superficial resemblance to steroid
is often used as a gap junction 2003; Li, J.,
However, there are some controversialcarcinogenesis
This study was designed to determine the impacts of CBX on MCF7and BT20
cell growth and transcriptomics level.
The structure of carbenoxolone (CBX)
and BT20 human breast cancer cell lines, Cell culture plates and flasks were obtained from National cell bank of Iran (Pasteur institute, Iran) and IWAKI, Japan respectively. RPMI1640 medium,
0.02%), TriDithiotheritol (DTT), first strand reverse transcriptase (RT) buffer, Moloney murine leukemia virus reverse
purchased from Sigma Aldrich Co., (Poole, UK). Agarose and Fetal bovine serum (FBS) were bought from Gibco, Invitrogen (Paisley, UK). Ribonuclease inhibitor was obtained from
AKT1, GAPDH phosphate dehydrogenase)
2009; Mamot and Rochlitz.
which mainly undergoes to cell proliferation, ) as well as
involvement in the metastasis, angiogenesis and . 2009; Camp
n regards to these miscellaneous biomoleculesit is believed that EGFR signal transduction could be
which may be prevent or
icholson 2003)oxopropoxy)-11
prototypic which mediated via Gap
induces apoptosis CBX is a semi-synthetic
obtained from Licorice root and demonstrates a superficial resemblance to steroid
is often used as a gap junction Li, J.,
However, there are some controversialcarcinogenesis(Cronier,
This study was designed to determine the impacts of CBX on MCF7and BT20
cell growth and transcriptomics level.
The structure of carbenoxolone (CBX).
and BT20 human breast cancer cell lines, Cell culture plates and flasks were obtained from National cell bank of Iran (Pasteur institute, Iran) and IWAKI, Japan respectively. RPMI1640 medium,
0.02%), Tri-ReagDithiotheritol (DTT), first strand reverse transcriptase (RT) buffer, Moloney murine leukemia virus reverse
purchased from Sigma Aldrich Co., (Poole, UK). Agarose and Fetal bovine serum (FBS) were bought from Gibco, Invitrogen (Paisley, UK). Ribonuclease inhibitor was obtained from
AKT1, GAPDH phosphate dehydrogenase), TP53,
2009; Afaq Mamot and Rochlitz.
which mainly undergoes to cell proliferation, as well as
involvement in the metastasis, angiogenesis and . 2009; Camp et al
n regards to these miscellaneous biomoleculesit is believed that EGFR signal transduction could be
which may be prevent or
icholson 2003). CBX 11-oxo
prototypic intervia Gap
induces apoptosis synthetic
obtained from Licorice root and demonstrates a superficial resemblance to steroid
is often used as a gap junction Li, J., et al
However, there are some controversial issues (Cronier,
This study was designed to determine the impacts of CBX on MCF7and BT20
cell growth and transcriptomics level.
and BT20 human breast cancer cell lines, Cell culture plates and flasks were obtained from National cell bank of Iran (Pasteur institute, Iran) and IWAKI, Japan respectively. RPMI1640 medium,
ReagDithiotheritol (DTT), first strand reverse transcriptase (RT) buffer, Moloney murine leukemia virus reverse
purchased from Sigma Aldrich Co., (Poole, UK). Agarose and Fetal bovine serum (FBS) were bought from Gibco, Invitrogen (Paisley, UK). Ribonuclease inhibitor was obtained from
AKT1, GAPDH , TP53,
Afaq Mamot and Rochlitz.
which mainly undergoes to cell proliferation, as well as
involvement in the metastasis, angiogenesis and et al.
n regards to these miscellaneous biomolecules, it is believed that EGFR signal transduction could be
which may be prevent or
CBX oxo-
inter-via Gap
induces apoptosis in synthetic
obtained from Licorice root and demonstrates a superficial resemblance to steroid
is often used as a gap junction et al. issues
(Cronier, This study was designed to
determine the impacts of CBX on MCF7and BT20 cell growth and
transcriptomics level.
and BT20 human breast cancer cell lines, Cell culture plates and flasks were obtained from National cell bank of Iran (Pasteur institute, Iran) and IWAKI, Japan respectively. RPMI1640 medium,
Reagent, Dithiotheritol (DTT), first strand reverse transcriptase (RT) buffer, Moloney murine leukemia virus reverse-
purchased from Sigma Aldrich Co., (Poole, UK). Agarose and Fetal bovine serum (FBS) were bought from Gibco, Invitrogen (Paisley, UK). Ribonuclease inhibitor was obtained from
AKT1, GAPDH , TP53,
Afaq Mamot and Rochlitz.
which mainly undergoes to cell proliferation, as well as
involvement in the metastasis, angiogenesis and . ,
it is believed that EGFR signal transduction could be which may be
prevent or CBX
--
via Gap in
synthetic obtained from Licorice root
and demonstrates a superficial resemblance to steroid is often used as a gap junction
. issues
(Cronier, This study was designed to
determine the impacts of CBX on MCF7and BT20 cell growth and
and BT20 human breast cancer cell lines, Cell culture plates and flasks were obtained from National cell bank of Iran (Pasteur institute, Iran) and IWAKI, Japan respectively. RPMI1640 medium,
ent, Dithiotheritol (DTT), first strand reverse transcriptase
-purchased from
Sigma Aldrich Co., (Poole, UK). Agarose and Fetal bovine serum (FBS) were bought from Gibco, Invitrogen (Paisley, UK). Ribonuclease inhibitor was obtained from
AKT1, GAPDH , TP53,
BCL2 (BForward and reverse oligonucleotide primers were provided from MWGThe Applied pyrocaPenicillIran). Taq DNA polymerase, polymerase chain reaction (PCR) reaction buffer, Random hexamer primers (pdN6), deoxynucleotide triphosphate monomers (dNTPs) and M(Crawley, UK). Carbenoxolone was obtained from Sigma Aldrich Co., (Poole, UK). All other chemicals not mentioned above were in the highest quality available.
Cell cultureThe MCF7and BT20 human breast cancer seededcells/cm2. These cells were saved in a humidified incubator (95% air and 5% CO2) at 37media consistFBS, penicillin G (200 U/ml) and streptomycin (200 concentrations 600lay open to expression profiling using real time RT
Cell The culturedat 40the 96CBX24 andgroup were washed once with phosphate buffered saline (PBS) and culture medium was replaced with 150 fresh media and then 50 PBS) was added to each well. After a 4 hr incubation with MTT at 37were exposed to 200 buffer (0.1 M glycine, 0.1 M NaCl, pH 10.5). The cultures were incubated for 30 min at 37crystals dissolvingmeasured at 570 nmreader, ELx 800 (Biotek, CA, USA).exclusion test was performed assessment of CBX. Briefly, cells were counted based onthe hemocytometedeath cell numbers. were conducted for morphological assessments using Olympus invert microscope Olympus DP20 camera and (Olympus optical Co.,
BCL2 (BForward and reverse oligonucleotide primers were provided from MWGThe SYBR Green PCR Master Mix Applied pyrocaPenicillIran). Taq DNA polymerase, polymerase chain reaction (PCR) reaction buffer, Random hexamer primers (pdN6), deoxynucleotide triphosphate monomers (dNTPs) and M(Crawley, UK). Carbenoxolone was obtained from Sigma Aldrich Co., (Poole, UK). All other chemicals not mentioned above were in the highest quality available.
Cell cultureThe MCF7and BT20 human breast cancer seededcells/cm2. These cells were saved in a humidified incubator (95% air and 5% CO2) at 37media consistFBS, penicillin G (200 U/ml) and streptomycin (200 g/ml). CBX was solved in RPMI 1640 media at concentrations 600lay open to expression profiling using real time RT
Cell vThe culturedat 40the 96CBX24 andgroup were washed once with phosphate buffered saline (PBS) and culture medium was replaced with 150 fresh media and then 50 PBS) was added to each well. After a 4 hr incubation with MTT at 37were exposed to 200 buffer (0.1 M glycine, 0.1 M NaCl, pH 10.5). The cultures were incubated for 30 min at 37crystals dissolvingmeasured at 570 nmreader, ELx 800 (Biotek, CA, USA).exclusion test was performed assessment of CBX. Briefly, cells were counted based onthe abilityhemocytometedeath cell numbers. were conducted for morphological assessments using Olympus invert microscope Olympus DP20 camera and (Olympus optical Co.,
BCL2 (BForward and reverse oligonucleotide primers were provided from MWG
SYBR Green PCR Master Mix Applied pyrocarbonate (DEPC) treated Penicillin G Iran). Taq DNA polymerase, polymerase chain reaction (PCR) reaction buffer, Random hexamer primers (pdN6), deoxynucleotide triphosphate monomers (dNTPs) and M(Crawley, UK). Carbenoxolone was obtained from Sigma Aldrich Co., (Poole, UK). All other chemicals not mentioned above were in the highest quality available.
Cell cultureThe MCF7and BT20 human breast cancer seeded cells/cm2. These cells were saved in a humidified incubator (95% air and 5% CO2) at 37media consistFBS, penicillin G (200 U/ml) and streptomycin (200
g/ml). CBX was solved in RPMI 1640 media at concentrations
M and finally 1000lay open to expression profiling using real time RT
viability The culturedat 40–50% confluency were subjected to MTT assay in the 96-well plates. The CBX concentrations24 and 48 hours at 37group were washed once with phosphate buffered saline (PBS) and culture medium was replaced with 150 fresh media and then 50 PBS) was added to each well. After a 4 hr incubation with MTT at 37were exposed to 200 buffer (0.1 M glycine, 0.1 M NaCl, pH 10.5). The cultures were incubated for 30 min at 37crystals dissolvingmeasured at 570 nmreader, ELx 800 (Biotek, CA, USA).exclusion test was performed assessment of CBX. Briefly, cells were counted based on
abilityhemocytometedeath cell numbers. were conducted for morphological assessments using Olympus invert microscope Olympus DP20 camera and (Olympus optical Co.,
Copyright © 2011 by Tabriz University of Medical Sciences
BCL2 (B-cell lymphoma 2)Forward and reverse oligonucleotide primers were provided from MWG
SYBR Green PCR Master Mix Applied Biosystems, Foster City, USA.
rbonate (DEPC) treated in G
Iran). Taq DNA polymerase, polymerase chain reaction (PCR) reaction buffer, Random hexamer primers (pdN6), deoxynucleotide triphosphate monomers (dNTPs) and M(Crawley, UK). Carbenoxolone was obtained from Sigma Aldrich Co., (Poole, UK). All other chemicals not mentioned above were in the highest quality available.
Cell cultureThe MCF7and BT20 human breast cancer
on 6cells/cm2. These cells were saved in a humidified incubator (95% air and 5% CO2) at 37media consistFBS, penicillin G (200 U/ml) and streptomycin (200
g/ml). CBX was solved in RPMI 1640 media at concentrations
M and finally 1000lay open to expression profiling using real time RT
iability The cultured
50% confluency were subjected to MTT assay in well plates. The
concentrations48 hours at 37
group were washed once with phosphate buffered saline (PBS) and culture medium was replaced with 150 fresh media and then 50 PBS) was added to each well. After a 4 hr incubation with MTT at 37were exposed to 200 buffer (0.1 M glycine, 0.1 M NaCl, pH 10.5). The cultures were incubated for 30 min at 37crystals dissolvingmeasured at 570 nmreader, ELx 800 (Biotek, CA, USA).exclusion test was performed assessment of CBX. Briefly, cells were counted based on
ability of cells to exclude trypan blue dye using a hemocytometedeath cell numbers. were conducted for morphological assessments using Olympus invert microscope Olympus DP20 camera and (Olympus optical Co.,
Copyright © 2011 by Tabriz University of Medical Sciences
cell lymphoma 2)Forward and reverse oligonucleotide primers were provided from MWG
SYBR Green PCR Master Mix Biosystems, Foster City, USA.
rbonate (DEPC) treated in G were obtained
Iran). Taq DNA polymerase, polymerase chain reaction (PCR) reaction buffer, Random hexamer primers (pdN6), deoxynucleotide triphosphate monomers (dNTPs) and M(Crawley, UK). Carbenoxolone was obtained from Sigma Aldrich Co., (Poole, UK). All other chemicals not mentioned above were in the highest quality available.
Cell culture The MCF7and BT20 human breast cancer
on 6-well plates at a density of 4cells/cm2. These cells were saved in a humidified incubator (95% air and 5% CO2) at 37media consistedFBS, penicillin G (200 U/ml) and streptomycin (200
g/ml). CBX was solved in RPMI 1640 media at concentrations
M and finally 1000lay open to viability examinations as well as gene expression profiling using real time RT
iability assesThe cultured MCF7and BT20 human breast cancer
50% confluency were subjected to MTT assay in well plates. The
concentrations48 hours at 37
group were washed once with phosphate buffered saline (PBS) and culture medium was replaced with 150 fresh media and then 50 PBS) was added to each well. After a 4 hr incubation with MTT at 37were exposed to 200 buffer (0.1 M glycine, 0.1 M NaCl, pH 10.5). The cultures were incubated for 30 min at 37crystals dissolvingmeasured at 570 nmreader, ELx 800 (Biotek, CA, USA).exclusion test was performed assessment of CBX. Briefly, cells were counted based on
of cells to exclude trypan blue dye using a hemocytometer for obtaining vital cell numbers versus death cell numbers. were conducted for morphological assessments using Olympus invert microscope Olympus DP20 camera and (Olympus optical Co.,
Copyright © 2011 by Tabriz University of Medical Sciences
cell lymphoma 2)Forward and reverse oligonucleotide primers were provided from MWG
SYBR Green PCR Master Mix Biosystems, Foster City, USA.
rbonate (DEPC) treated were obtained
Iran). Taq DNA polymerase, polymerase chain reaction (PCR) reaction buffer, Random hexamer primers (pdN6), deoxynucleotide triphosphate monomers (dNTPs) and MgCl2 were(Crawley, UK). Carbenoxolone was obtained from Sigma Aldrich Co., (Poole, UK). All other chemicals not mentioned above were in the highest quality available.
The MCF7and BT20 human breast cancer well plates at a density of 4
cells/cm2. These cells were saved in a humidified incubator (95% air and 5% CO2) at 37
ed of RPMI 1640 complemented with 10% FBS, penicillin G (200 U/ml) and streptomycin (200
g/ml). CBX was solved in RPMI 1640 media at concentrations of 50
M and finally 1000viability examinations as well as gene
expression profiling using real time RT
ssessment MCF7and BT20 human breast cancer
50% confluency were subjected to MTT assay in well plates. The
concentrations48 hours at 37
group were washed once with phosphate buffered saline (PBS) and culture medium was replaced with 150 fresh media and then 50 PBS) was added to each well. After a 4 hr incubation with MTT at 37˚C, medium was removed and the cells were exposed to 200 buffer (0.1 M glycine, 0.1 M NaCl, pH 10.5). The cultures were incubated for 30 min at 37crystals dissolvingmeasured at 570 nmreader, ELx 800 (Biotek, CA, USA).exclusion test was performed assessment of CBX. Briefly, cells were counted based on
of cells to exclude trypan blue dye using a r for obtaining vital cell numbers versus
death cell numbers. were conducted for morphological assessments using Olympus invert microscope Olympus DP20 camera and (Olympus optical Co.,
Copyright © 2011 by Tabriz University of Medical Sciences
cell lymphoma 2)Forward and reverse oligonucleotide primers were provided from MWG-
SYBR Green PCR Master Mix Biosystems, Foster City, USA.
rbonate (DEPC) treated were obtained
Iran). Taq DNA polymerase, polymerase chain reaction (PCR) reaction buffer, Random hexamer primers (pdN6), deoxynucleotide triphosphate monomers
gCl2 were(Crawley, UK). Carbenoxolone was obtained from Sigma Aldrich Co., (Poole, UK). All other chemicals not mentioned above were in the highest quality available.
The MCF7and BT20 human breast cancer well plates at a density of 4
cells/cm2. These cells were saved in a humidified incubator (95% air and 5% CO2) at 37
of RPMI 1640 complemented with 10% FBS, penicillin G (200 U/ml) and streptomycin (200
g/ml). CBX was solved in RPMI 1640 media at of 50
M and finally 1000viability examinations as well as gene
expression profiling using real time RT
sment MCF7and BT20 human breast cancer
50% confluency were subjected to MTT assay in well plates. The
concentrations and 48 hours at 37
group were washed once with phosphate buffered saline (PBS) and culture medium was replaced with 150 fresh media and then 50 PBS) was added to each well. After a 4 hr incubation
C, medium was removed and the cells were exposed to 200 buffer (0.1 M glycine, 0.1 M NaCl, pH 10.5). The cultures were incubated for 30 min at 37crystals dissolving and then UV absorbance was measured at 570 nm reader, ELx 800 (Biotek, CA, USA).exclusion test was performed assessment of CBX. Briefly, cells were counted based on
of cells to exclude trypan blue dye using a r for obtaining vital cell numbers versus
death cell numbers. were conducted for morphological assessments using Olympus invert microscope Olympus DP20 camera and (Olympus optical Co.,
Copyright © 2011 by Tabriz University of Medical Sciences
cell lymphoma 2)Forward and reverse oligonucleotide primers were
-Europhin, (Ebersberg, Germany).SYBR Green PCR Master Mix
Biosystems, Foster City, USA. rbonate (DEPC) treated
were obtained Iran). Taq DNA polymerase, polymerase chain reaction (PCR) reaction buffer, Random hexamer primers (pdN6), deoxynucleotide triphosphate monomers
gCl2 were(Crawley, UK). Carbenoxolone was obtained from Sigma Aldrich Co., (Poole, UK). All other chemicals not mentioned above were in the highest quality available.
The MCF7and BT20 human breast cancer well plates at a density of 4
cells/cm2. These cells were saved in a humidified incubator (95% air and 5% CO2) at 37
of RPMI 1640 complemented with 10% FBS, penicillin G (200 U/ml) and streptomycin (200
g/ml). CBX was solved in RPMI 1640 media at of 50M, 100
M and finally 1000viability examinations as well as gene
expression profiling using real time RT
sment MCF7and BT20 human breast cancer
50% confluency were subjected to MTT assay in well plates. The cells were treated with a range of
and each group were 48 hours at 37˚C. After which, the cells of each
group were washed once with phosphate buffered saline (PBS) and culture medium was replaced with 150 fresh media and then 50 PBS) was added to each well. After a 4 hr incubation
C, medium was removed and the cells l DMSO and
buffer (0.1 M glycine, 0.1 M NaCl, pH 10.5). The cultures were incubated for 30 min at 37
and then UV absorbance was using a spectrophotometric plate
reader, ELx 800 (Biotek, CA, USA).exclusion test was performed assessment of CBX. Briefly, cells were counted based on
of cells to exclude trypan blue dye using a r for obtaining vital cell numbers versus
death cell numbers. Light microscopic examinations were conducted for morphological assessments using Olympus invert microscope Olympus DP20 camera and (Olympus optical Co., Ltd., Tokyo, Japan)
Copyright © 2011 by Tabriz University of Medical Sciences
cell lymphoma 2) andForward and reverse oligonucleotide primers were
Europhin, (Ebersberg, Germany).SYBR Green PCR Master Mix
Biosystems, Foster City, USA. rbonate (DEPC) treated
were obtained Iran). Taq DNA polymerase, polymerase chain reaction (PCR) reaction buffer, Random hexamer primers (pdN6), deoxynucleotide triphosphate monomers
gCl2 were (Crawley, UK). Carbenoxolone was obtained from Sigma Aldrich Co., (Poole, UK). All other chemicals not mentioned above were in the highest quality available.
The MCF7and BT20 human breast cancer well plates at a density of 4
cells/cm2. These cells were saved in a humidified incubator (95% air and 5% CO2) at 37
of RPMI 1640 complemented with 10% FBS, penicillin G (200 U/ml) and streptomycin (200
g/ml). CBX was solved in RPMI 1640 media at M, 100M (1Mm)
viability examinations as well as gene expression profiling using real time RT
MCF7and BT20 human breast cancer
50% confluency were subjected to MTT assay in cells were treated with a range of
each group were . After which, the cells of each
group were washed once with phosphate buffered saline (PBS) and culture medium was replaced with 150 fresh media and then 50 l MTT reagent (2 mg/ml in PBS) was added to each well. After a 4 hr incubation
C, medium was removed and the cells l DMSO and
buffer (0.1 M glycine, 0.1 M NaCl, pH 10.5). The cultures were incubated for 30 min at 37
and then UV absorbance was using a spectrophotometric plate
reader, ELx 800 (Biotek, CA, USA).exclusion test was performed assessment of CBX. Briefly, cells were counted based on
of cells to exclude trypan blue dye using a r for obtaining vital cell numbers versus
Light microscopic examinations were conducted for morphological assessments using Olympus invert microscope Olympus DP20 camera and
Ltd., Tokyo, Japan)
Copyright © 2011 by Tabriz University of Medical Sciences
and Forward and reverse oligonucleotide primers were
Europhin, (Ebersberg, Germany).SYBR Green PCR Master Mix
Biosystems, Foster City, USA. rbonate (DEPC) treated water; streptomycin and
were obtained from Iran). Taq DNA polymerase, polymerase chain reaction (PCR) reaction buffer, Random hexamer primers (pdN6), deoxynucleotide triphosphate monomers
obtained(Crawley, UK). Carbenoxolone was obtained from Sigma Aldrich Co., (Poole, UK). All other chemicals not mentioned above were in the highest quality available.
The MCF7and BT20 human breast cancer well plates at a density of 4
cells/cm2. These cells were saved in a humidified incubator (95% air and 5% CO2) at 37
of RPMI 1640 complemented with 10% FBS, penicillin G (200 U/ml) and streptomycin (200
g/ml). CBX was solved in RPMI 1640 media at M, 100
M (1Mm) viability examinations as well as gene
expression profiling using real time RT
MCF7and BT20 human breast cancer 50% confluency were subjected to MTT assay in
cells were treated with a range of each group were
. After which, the cells of each group were washed once with phosphate buffered saline (PBS) and culture medium was replaced with 150
l MTT reagent (2 mg/ml in PBS) was added to each well. After a 4 hr incubation
C, medium was removed and the cells l DMSO and
buffer (0.1 M glycine, 0.1 M NaCl, pH 10.5). The cultures were incubated for 30 min at 37
and then UV absorbance was using a spectrophotometric plate
reader, ELx 800 (Biotek, CA, USA).exclusion test was performed assessment of CBX. Briefly, cells were counted based on
of cells to exclude trypan blue dye using a r for obtaining vital cell numbers versus
Light microscopic examinations were conducted for morphological assessments using Olympus invert microscope CKX41 equipped with Olympus DP20 camera and
Ltd., Tokyo, Japan)
Copyright © 2011 by Tabriz University of Medical Sciences
CYCS (Cytochrome c)Forward and reverse oligonucleotide primers were
Europhin, (Ebersberg, Germany).SYBR Green PCR Master Mix
Biosystems, Foster City, USA. water; streptomycin and
from CinnaGen, (Tehran, Iran). Taq DNA polymerase, polymerase chain reaction (PCR) reaction buffer, Random hexamer primers (pdN6), deoxynucleotide triphosphate monomers
obtained(Crawley, UK). Carbenoxolone was obtained from Sigma Aldrich Co., (Poole, UK). All other chemicals not mentioned above were in the highest quality available.
The MCF7and BT20 human breast cancer well plates at a density of 4
cells/cm2. These cells were saved in a humidified incubator (95% air and 5% CO2) at 37
of RPMI 1640 complemented with 10% FBS, penicillin G (200 U/ml) and streptomycin (200
g/ml). CBX was solved in RPMI 1640 media at M, 150
M (1Mm) viability examinations as well as gene
expression profiling using real time RT
MCF7and BT20 human breast cancer 50% confluency were subjected to MTT assay in
cells were treated with a range of each group were
. After which, the cells of each group were washed once with phosphate buffered saline (PBS) and culture medium was replaced with 150
l MTT reagent (2 mg/ml in PBS) was added to each well. After a 4 hr incubation
C, medium was removed and the cells l DMSO and
buffer (0.1 M glycine, 0.1 M NaCl, pH 10.5). The cultures were incubated for 30 min at 37
and then UV absorbance was using a spectrophotometric plate
reader, ELx 800 (Biotek, CA, USA).exclusion test was performed for growth inhibition assessment of CBX. Briefly, cells were counted based on
of cells to exclude trypan blue dye using a r for obtaining vital cell numbers versus
Light microscopic examinations were conducted for morphological assessments using
CKX41 equipped with Olympus DP20 camera and CellA 3.3 software
Ltd., Tokyo, Japan)
Copyright © 2011 by Tabriz University of Medical Sciences
CYCS (Cytochrome c)Forward and reverse oligonucleotide primers were
Europhin, (Ebersberg, Germany).SYBR Green PCR Master Mix was obtained from
Biosystems, Foster City, USA. water; streptomycin and
CinnaGen, (Tehran, Iran). Taq DNA polymerase, polymerase chain reaction (PCR) reaction buffer, Random hexamer primers (pdN6), deoxynucleotide triphosphate monomers
obtained (Crawley, UK). Carbenoxolone was obtained from Sigma Aldrich Co., (Poole, UK). All other chemicals not mentioned above were in the highest quality available.
The MCF7and BT20 human breast cancer well plates at a density of 4
cells/cm2. These cells were saved in a humidified incubator (95% air and 5% CO2) at 37
of RPMI 1640 complemented with 10% FBS, penicillin G (200 U/ml) and streptomycin (200
g/ml). CBX was solved in RPMI 1640 media at M, 150
M (1Mm) and viability examinations as well as gene
expression profiling using real time RT-PCR.
MCF7and BT20 human breast cancer 50% confluency were subjected to MTT assay in
cells were treated with a range of each group were
. After which, the cells of each group were washed once with phosphate buffered saline (PBS) and culture medium was replaced with 150
l MTT reagent (2 mg/ml in PBS) was added to each well. After a 4 hr incubation
C, medium was removed and the cells l DMSO and 25
buffer (0.1 M glycine, 0.1 M NaCl, pH 10.5). The cultures were incubated for 30 min at 37
and then UV absorbance was using a spectrophotometric plate
reader, ELx 800 (Biotek, CA, USA).for growth inhibition
assessment of CBX. Briefly, cells were counted based onof cells to exclude trypan blue dye using a
r for obtaining vital cell numbers versus Light microscopic examinations
were conducted for morphological assessments using CKX41 equipped with
CellA 3.3 software Ltd., Tokyo, Japan)
Copyright © 2011 by Tabriz University of Medical Sciences
CYCS (Cytochrome c)Forward and reverse oligonucleotide primers were
Europhin, (Ebersberg, Germany).was obtained from
Biosystems, Foster City, USA. water; streptomycin and
CinnaGen, (Tehran, Iran). Taq DNA polymerase, polymerase chain reaction (PCR) reaction buffer, Random hexamer primers (pdN6), deoxynucleotide triphosphate monomers
from QIAGEN (Crawley, UK). Carbenoxolone was obtained from Sigma Aldrich Co., (Poole, UK). All other chemicals not mentioned above were in the highest quality available.
The MCF7and BT20 human breast cancer well plates at a density of 4
cells/cm2. These cells were saved in a humidified incubator (95% air and 5% CO2) at 37˚C. Cell culture
of RPMI 1640 complemented with 10% FBS, penicillin G (200 U/ml) and streptomycin (200
g/ml). CBX was solved in RPMI 1640 media at M, 150
and then viability examinations as well as gene
PCR.
MCF7and BT20 human breast cancer 50% confluency were subjected to MTT assay in
cells were treated with a range of each group were incubated for
. After which, the cells of each group were washed once with phosphate buffered saline (PBS) and culture medium was replaced with 150
l MTT reagent (2 mg/ml in PBS) was added to each well. After a 4 hr incubation
C, medium was removed and the cells 5 l of Sorenson
buffer (0.1 M glycine, 0.1 M NaCl, pH 10.5). The cultures were incubated for 30 min at 37˚C
and then UV absorbance was using a spectrophotometric plate
reader, ELx 800 (Biotek, CA, USA). Trypan blue for growth inhibition
assessment of CBX. Briefly, cells were counted based onof cells to exclude trypan blue dye using a
r for obtaining vital cell numbers versus Light microscopic examinations
were conducted for morphological assessments using CKX41 equipped with
CellA 3.3 software Ltd., Tokyo, Japan).
Copyright © 2011 by Tabriz University of Medical Sciences
CYCS (Cytochrome c)Forward and reverse oligonucleotide primers were
Europhin, (Ebersberg, Germany).was obtained from
Biosystems, Foster City, USA. water; streptomycin and
CinnaGen, (Tehran, Iran). Taq DNA polymerase, polymerase chain reaction (PCR) reaction buffer, Random hexamer primers (pdN6), deoxynucleotide triphosphate monomers
from QIAGEN (Crawley, UK). Carbenoxolone was obtained from Sigma Aldrich Co., (Poole, UK). All other chemicals not mentioned above were in the highest quality available.
The MCF7and BT20 human breast cancer cells were well plates at a density of 4
cells/cm2. These cells were saved in a humidified ˚C. Cell culture
of RPMI 1640 complemented with 10% FBS, penicillin G (200 U/ml) and streptomycin (200
g/ml). CBX was solved in RPMI 1640 media at M, 300
then cells were viability examinations as well as gene
PCR.
MCF7and BT20 human breast cancer 50% confluency were subjected to MTT assay in
cells were treated with a range of incubated for
. After which, the cells of each group were washed once with phosphate buffered saline (PBS) and culture medium was replaced with 150
l MTT reagent (2 mg/ml in PBS) was added to each well. After a 4 hr incubation
C, medium was removed and the cells l of Sorenson
buffer (0.1 M glycine, 0.1 M NaCl, pH 10.5). The for formazan
and then UV absorbance was using a spectrophotometric plate
Trypan blue for growth inhibition
assessment of CBX. Briefly, cells were counted based onof cells to exclude trypan blue dye using a
r for obtaining vital cell numbers versus Light microscopic examinations
were conducted for morphological assessments using CKX41 equipped with
CellA 3.3 software
Copyright © 2011 by Tabriz University of Medical Sciences
CYCS (Cytochrome c)Forward and reverse oligonucleotide primers were
Europhin, (Ebersberg, Germany).was obtained from
Biosystems, Foster City, USA. Diethyl water; streptomycin and
CinnaGen, (Tehran, Iran). Taq DNA polymerase, polymerase chain reaction (PCR) reaction buffer, Random hexamer primers (pdN6), deoxynucleotide triphosphate monomers
from QIAGEN (Crawley, UK). Carbenoxolone was obtained from Sigma Aldrich Co., (Poole, UK). All other chemicals not mentioned above were in the highest quality available.
cells were well plates at a density of 4.
cells/cm2. These cells were saved in a humidified ˚C. Cell culture
of RPMI 1640 complemented with 10% FBS, penicillin G (200 U/ml) and streptomycin (200
g/ml). CBX was solved in RPMI 1640 media at M, 300
cells were viability examinations as well as gene
MCF7and BT20 human breast cancer 50% confluency were subjected to MTT assay in
cells were treated with a range of incubated for
. After which, the cells of each group were washed once with phosphate buffered saline (PBS) and culture medium was replaced with 150
l MTT reagent (2 mg/ml in PBS) was added to each well. After a 4 hr incubation
C, medium was removed and the cells l of Sorenson
buffer (0.1 M glycine, 0.1 M NaCl, pH 10.5). The for formazan
and then UV absorbance was using a spectrophotometric plate
Trypan blue for growth inhibition
assessment of CBX. Briefly, cells were counted based onof cells to exclude trypan blue dye using a
r for obtaining vital cell numbers versus Light microscopic examinations
were conducted for morphological assessments using CKX41 equipped with
CellA 3.3 software
Copyright © 2011 by Tabriz University of Medical Sciences
CYCS (Cytochrome c)Forward and reverse oligonucleotide primers were
Europhin, (Ebersberg, Germany).was obtained from
Diethyl water; streptomycin and
CinnaGen, (Tehran, Iran). Taq DNA polymerase, polymerase chain reaction (PCR) reaction buffer, Random hexamer primers (pdN6), deoxynucleotide triphosphate monomers
from QIAGEN (Crawley, UK). Carbenoxolone was obtained from Sigma Aldrich Co., (Poole, UK). All other chemicals not mentioned above were in the highest quality available.
cells were .0×10
cells/cm2. These cells were saved in a humidified ˚C. Cell culture
of RPMI 1640 complemented with 10% FBS, penicillin G (200 U/ml) and streptomycin (200
g/ml). CBX was solved in RPMI 1640 media at M, 300M,
cells were viability examinations as well as gene
MCF7and BT20 human breast cancer cells 50% confluency were subjected to MTT assay in
cells were treated with a range of incubated for
. After which, the cells of each group were washed once with phosphate buffered saline (PBS) and culture medium was replaced with 150
l MTT reagent (2 mg/ml in PBS) was added to each well. After a 4 hr incubation
C, medium was removed and the cells l of Sorenson
buffer (0.1 M glycine, 0.1 M NaCl, pH 10.5). The for formazan
and then UV absorbance was using a spectrophotometric plate
Trypan blue for growth inhibition
assessment of CBX. Briefly, cells were counted based onof cells to exclude trypan blue dye using a
r for obtaining vital cell numbers versus Light microscopic examinations
were conducted for morphological assessments using CKX41 equipped with
CellA 3.3 software
Copyright © 2011 by Tabriz University of Medical Sciences
CYCS (Cytochrome c) Forward and reverse oligonucleotide primers were
Europhin, (Ebersberg, Germany). was obtained from
Diethyl water; streptomycin and
CinnaGen, (Tehran, Iran). Taq DNA polymerase, polymerase chain reaction (PCR) reaction buffer, Random hexamer primers (pdN6), deoxynucleotide triphosphate monomers
from QIAGEN (Crawley, UK). Carbenoxolone was obtained from Sigma Aldrich Co., (Poole, UK). All other chemicals not
cells were 0×104
cells/cm2. These cells were saved in a humidified ˚C. Cell culture
of RPMI 1640 complemented with 10% FBS, penicillin G (200 U/ml) and streptomycin (200
g/ml). CBX was solved in RPMI 1640 media at M,
cells were viability examinations as well as gene
cells 50% confluency were subjected to MTT assay in
cells were treated with a range of incubated for
. After which, the cells of each group were washed once with phosphate buffered saline (PBS) and culture medium was replaced with 150 l
l MTT reagent (2 mg/ml in PBS) was added to each well. After a 4 hr incubation
C, medium was removed and the cells l of Sorenson
buffer (0.1 M glycine, 0.1 M NaCl, pH 10.5). The for formazan
and then UV absorbance was using a spectrophotometric plate
Trypan blue for growth inhibition
assessment of CBX. Briefly, cells were counted based on of cells to exclude trypan blue dye using a
r for obtaining vital cell numbers versus Light microscopic examinations
were conducted for morphological assessments using CKX41 equipped with
CellA 3.3 software
Gap junctions in cancer cells
Copyright © 2011 by Tabriz University of Medical Sciences
Gene expression Cells were subjected to gene expression evaluation justat 150exposure TriReagent quality and respectively by electrophoresis and NanoDrop®Spectrophotometer (NanoWilmington, DE, published Gene Bank 5.01 http://www.premierbiosoft.com) and listed in Table 1.The reverse transcripti11 DTT, 2 g of RN(200 U) in a total volume of 20 program was: 95 and reverse transcriptase, and incubation at 25 min, 42 real time PCR examinations. PCR experiment was performed to assess the expression of selected key genes as mentioned above.All amplification reactions were performed in a total 25µl volume using the iQ5 Optical System (BioLaboratories I1 µl cDNA, 1 µl primer (100 nM each primer), 12.5 µl 2X Power SYBR Green PCR Master Mix and 10.5 µl RNAse/DNAse free water. Thermal cycling conditions were as follow95˚C Interpretation of the result was performed using the Pfaffle method and the CT values were normalized to the expression rate of (Pfaffl 2001and negative control included in each experimentas internal positive co
Data analyzingOne way ANOVA was performed for statistical analysis using SPSS version 18 following with
Gap junctions in cancer cells
Copyright © 2011 by Tabriz University of Medical Sciences
Gene expression Cells were subjected to gene expression evaluation justat 150exposure TriReagent quality and respectively by electrophoresis and NanoDrop®Spectrophotometer (NanoWilmington, DE, published Gene Bank 5.01 http://www.premierbiosoft.com) and listed in Table 1.The reverse transcripti11 l DEPC treated water, 4 DTT, 2
g of RN(200 U) in a total volume of 20 program was: 95 and reverse transcriptase, and incubation at 25 min, 42 real time PCR examinations. PCR experiment was performed to assess the expression of selected key genes as mentioned above.All amplification reactions were performed in a total 25µl volume using the iQ5 Optical System (BioLaboratories I1 µl cDNA, 1 µl primer (100 nM each primer), 12.5 µl 2X Power SYBR Green PCR Master Mix and 10.5 µl RNAse/DNAse free water. Thermal cycling conditions were as follow
˚C for 15 sec, 56Interpretation of the result was performed using the Pfaffle method and the CT values were normalized to the expression rate of Pfaffl 2001
and negative control included in each experimentas internal positive co
Data analyzingOne way ANOVA was performed for statistical analysis using SPSS version 18 following with
Gap junctions in cancer cells
Copyright © 2011 by Tabriz University of Medical Sciences
Gene expression Cells were subjected to gene expression evaluation just
M which exposure time of 24 hr andTriReagent quality and respectively by electrophoresis and NanoDrop®Spectrophotometer (NanoWilmington, DE, published Gene Bank 5.01 http://www.premierbiosoft.com) and listed in Table 1.The reverse transcripti
DEPC treated water, 4 DTT, 2 l
g of RNA, 0.5 (200 U) in a total volume of 20 program was: 95 and reverse transcriptase, and incubation at 25 min, 42 ˚C for 50 min and then they were real time PCR examinations. PCR experiment was performed to assess the expression of selected key genes as mentioned above.All amplification reactions were performed in a total 25µl volume using the iQ5 Optical System (BioLaboratories I1 µl cDNA, 1 µl primer (100 nM each primer), 12.5 µl 2X Power SYBR Green PCR Master Mix and 10.5 µl RNAse/DNAse free water. Thermal cycling conditions were as follow
for 15 sec, 56Interpretation of the result was performed using the Pfaffle method and the CT values were normalized to the expression rate of Pfaffl 2001
and negative control included in each experimentas internal positive co
Data analyzingOne way ANOVA was performed for statistical analysis using SPSS version 18 following with
Gap junctions in cancer cells
Copyright © 2011 by Tabriz University of Medical Sciences
Gene expression Cells were subjected to gene expression evaluation just
M which time of 24 hr and
TriReagent accordingquality and quantity ofrespectively by electrophoresis and NanoDrop®Spectrophotometer (NanoWilmington, DE, published Gene Bank 5.01 (Premier Biosoft International, http://www.premierbiosoft.com) and listed in Table 1.The reverse transcripti
DEPC treated water, 4 dNTPs (10 mM), 0.5 A, 0.5
(200 U) in a total volume of 20 program was: 95 and reverse transcriptase, and incubation at 25
C for 50 min and then they were real time PCR examinations. PCR experiment was performed to assess the expression of selected key genes as mentioned above.All amplification reactions were performed in a total 25µl volume using the iQ5 Optical System (BioLaboratories I1 µl cDNA, 1 µl primer (100 nM each primer), 12.5 µl 2X Power SYBR Green PCR Master Mix and 10.5 µl RNAse/DNAse free water. Thermal cycling conditions were as follow
for 15 sec, 56Interpretation of the result was performed using the Pfaffle method and the CT values were normalized to the expression rate of Pfaffl 2001). All reactions were performed in triplicate
and negative control included in each experimentas internal positive co
Data analyzingOne way ANOVA was performed for statistical analysis using SPSS version 18 following with
Gap junctions in cancer cells
Copyright © 2011 by Tabriz University of Medical Sciences
Gene expression analysisCells were subjected to gene expression evaluation just
M which time of 24 hr and
accordingquantity of
respectively by electrophoresis and NanoDrop®Spectrophotometer (NanoWilmington, DE, published Gene Bank
(Premier Biosoft International, http://www.premierbiosoft.com) and listed in Table 1.The reverse transcripti
DEPC treated water, 4 dNTPs (10 mM), 0.5
A, 0.5 (200 U) in a total volume of 20 program was: 95 ˚and reverse transcriptase, and incubation at 25
C for 50 min and then they were real time PCR examinations. PCR experiment was performed to assess the expression of selected key genes as mentioned above.All amplification reactions were performed in a total 25µl volume using the iQ5 Optical System (BioLaboratories Inc., Hercules, USA). Each well contained: 1 µl cDNA, 1 µl primer (100 nM each primer), 12.5 µl 2X Power SYBR Green PCR Master Mix and 10.5 µl RNAse/DNAse free water. Thermal cycling conditions were as follows: 1 cycle at 94
for 15 sec, 56Interpretation of the result was performed using the Pfaffle method and the CT values were normalized to the expression rate of
. All reactions were performed in triplicate and negative control included in each experimentas internal positive co
Data analyzing One way ANOVA was performed for statistical analysis using SPSS version 18 following with
Gap junctions in cancer cells
Copyright © 2011 by Tabriz University of Medical Sciences
analysisCells were subjected to gene expression evaluation just
M which was time of 24 hr and
accordingquantity of
respectively by electrophoresis and NanoDrop®Spectrophotometer (NanoWilmington, DE, USA). published Gene Bank
(Premier Biosoft International, http://www.premierbiosoft.com) and listed in Table 1.The reverse transcripti
DEPC treated water, 4 dNTPs (10 mM), 0.5
l RNasin (40 U), and 1 (200 U) in a total volume of 20
˚C for 5 min before addition of RNasin and reverse transcriptase, and incubation at 25
C for 50 min and then they were real time PCR examinations. PCR experiment was performed to assess the expression of selected key genes as mentioned above. All amplification reactions were performed in a total 25µl volume using the iQ5 Optical System (Bio
nc., Hercules, USA). Each well contained: 1 µl cDNA, 1 µl primer (100 nM each primer), 12.5 µl 2X Power SYBR Green PCR Master Mix and 10.5 µl RNAse/DNAse free water. Thermal cycling conditions
: 1 cycle at 94for 15 sec, 56-62
Interpretation of the result was performed using the Pfaffle method and the CT values were normalized to the expression rate of GAPDH
. All reactions were performed in triplicate and negative control included in each experimentas internal positive co
One way ANOVA was performed for statistical analysis using SPSS version 18 following with
Gene name
GAPDH
BCL2
AKT1
CYCS
STAT5
TP53
F: Forward; R:
Gap junctions in cancer cells
Copyright © 2011 by Tabriz University of Medical Sciences
analysisCells were subjected to gene expression evaluation just
was obtained as IC50 of CBX time of 24 hr and
according to manufacturer guideline. quantity of the extracted RNA was measured
respectively by electrophoresis and NanoDrop®Spectrophotometer (Nano
USA). published Gene Bank sequences using Beacon Designer
(Premier Biosoft International, http://www.premierbiosoft.com) and listed in Table 1.The reverse transcription reaction was performed using
DEPC treated water, 4 dNTPs (10 mM), 0.5
RNasin (40 U), and 1 (200 U) in a total volume of 20
C for 5 min before addition of RNasin and reverse transcriptase, and incubation at 25
C for 50 min and then they were real time PCR examinations. PCR experiment was performed to assess the expression of selected key genes
All amplification reactions were performed in a total 25µl volume using the iQ5 Optical System (Bio
nc., Hercules, USA). Each well contained: 1 µl cDNA, 1 µl primer (100 nM each primer), 12.5 µl 2X Power SYBR Green PCR Master Mix and 10.5 µl RNAse/DNAse free water. Thermal cycling conditions
: 1 cycle at 9462˚C for 30 sec, and 72˚C for 25 sec.
Interpretation of the result was performed using the Pfaffle method and the CT values were normalized to the
GAPDH. All reactions were performed in triplicate
and negative control included in each experimentas internal positive control
One way ANOVA was performed for statistical analysis using SPSS version 18 following with
Table 1.
Gene name
GAPDH
BCL2
AKT1
CYCS
STAT5
TP53
orward; R:
Copyright © 2011 by Tabriz University of Medical Sciences
analysis Cells were subjected to gene expression evaluation just
obtained as IC50 of CBX time of 24 hr and total RNA was isolated using
to manufacturer guideline. the extracted RNA was measured
respectively by electrophoresis and NanoDrop®Spectrophotometer (Nano
USA). Primers were designed from sequences using Beacon Designer
(Premier Biosoft International, http://www.premierbiosoft.com) and listed in Table 1.
on reaction was performed using DEPC treated water, 4
dNTPs (10 mM), 0.5 RNasin (40 U), and 1
(200 U) in a total volume of 20 C for 5 min before addition of RNasin
and reverse transcriptase, and incubation at 25 C for 50 min and then they were
real time PCR examinations. PCR experiment was performed to assess the expression of selected key genes
All amplification reactions were performed in a total 25µl volume using the iQ5 Optical System (Bio
nc., Hercules, USA). Each well contained: 1 µl cDNA, 1 µl primer (100 nM each primer), 12.5 µl 2X Power SYBR Green PCR Master Mix and 10.5 µl RNAse/DNAse free water. Thermal cycling conditions
: 1 cycle at 94˚C for 30 sec, and 72˚C for 25 sec.
Interpretation of the result was performed using the Pfaffle method and the CT values were normalized to the
GAPDH. All reactions were performed in triplicate
and negative control included in each experimentntrol.
One way ANOVA was performed for statistical analysis using SPSS version 18 following with
Table 1.
Gene name
orward; R:
Copyright © 2011 by Tabriz University of Medical Sciences
Cells were subjected to gene expression evaluation justobtained as IC50 of CBX
total RNA was isolated using to manufacturer guideline. the extracted RNA was measured
respectively by electrophoresis and NanoDrop®Spectrophotometer (Nano-
Primers were designed from sequences using Beacon Designer
(Premier Biosoft International, http://www.premierbiosoft.com) and listed in Table 1.
on reaction was performed using DEPC treated water, 4
dNTPs (10 mM), 0.5 RNasin (40 U), and 1
(200 U) in a total volume of 20 C for 5 min before addition of RNasin
and reverse transcriptase, and incubation at 25 C for 50 min and then they were
real time PCR examinations. PCR experiment was performed to assess the expression of selected key genes
All amplification reactions were performed in a total 25µl volume using the iQ5 Optical System (Bio
nc., Hercules, USA). Each well contained: 1 µl cDNA, 1 µl primer (100 nM each primer), 12.5 µl 2X Power SYBR Green PCR Master Mix and 10.5 µl RNAse/DNAse free water. Thermal cycling conditions
: 1 cycle at 94˚C for 10 min, 40 cycles at ˚C for 30 sec, and 72˚C for 25 sec.
Interpretation of the result was performed using the Pfaffle method and the CT values were normalized to the
GAPDH as a housekeeping gene. All reactions were performed in triplicate
and negative control included in each experiment
One way ANOVA was performed for statistical analysis using SPSS version 18 following with
Table 1.
orward; R: Reverse
Copyright © 2011 by Tabriz University of Medical Sciences
Cells were subjected to gene expression evaluation justobtained as IC50 of CBX
total RNA was isolated using to manufacturer guideline. the extracted RNA was measured
respectively by electrophoresis and NanoDrop® Dro
Primers were designed from sequences using Beacon Designer
(Premier Biosoft International, http://www.premierbiosoft.com) and listed in Table 1.
on reaction was performed using l MMLV buffered with
dNTPs (10 mM), 0.5 lRNasin (40 U), and 1
(200 U) in a total volume of 20 C for 5 min before addition of RNasin
and reverse transcriptase, and incubation at 25 C for 50 min and then they were
real time PCR examinations. PCR experiment was performed to assess the expression of selected key genes
All amplification reactions were performed in a total 25µl volume using the iQ5 Optical System (Bio
nc., Hercules, USA). Each well contained: 1 µl cDNA, 1 µl primer (100 nM each primer), 12.5 µl 2X Power SYBR Green PCR Master Mix and 10.5 µl RNAse/DNAse free water. Thermal cycling conditions
˚C for 10 min, 40 cycles at ˚C for 30 sec, and 72˚C for 25 sec.
Interpretation of the result was performed using the Pfaffle method and the CT values were normalized to the
as a housekeeping gene. All reactions were performed in triplicate
and negative control included in each experiment
One way ANOVA was performed for statistical analysis using SPSS version 18 following with
Table 1. Genes with corresponding primers which used for gene expression
Accession No.
NM_002046.3
NM_000633.2
NM_005163.2
NM_018947.4
NM_007315.3
NM_001126114
Reverse
Copyright © 2011 by Tabriz University of Medical Sciences
Cells were subjected to gene expression evaluation justobtained as IC50 of CBX
total RNA was isolated using to manufacturer guideline. the extracted RNA was measured
respectively by electrophoresis and NanoDrop®Drop Technologies,
Primers were designed from sequences using Beacon Designer
(Premier Biosoft International, http://www.premierbiosoft.com) and listed in Table 1.
on reaction was performed using MMLV buffered with l pdN6 (400 ng/
RNasin (40 U), and 1 (200 U) in a total volume of 20 l
C for 5 min before addition of RNasin and reverse transcriptase, and incubation at 25
C for 50 min and then they were real time PCR examinations. PCR experiment was performed to assess the expression of selected key genes
All amplification reactions were performed in a total 25µl volume using the iQ5 Optical System (Bio
nc., Hercules, USA). Each well contained: 1 µl cDNA, 1 µl primer (100 nM each primer), 12.5 µl 2X Power SYBR Green PCR Master Mix and 10.5 µl RNAse/DNAse free water. Thermal cycling conditions
˚C for 10 min, 40 cycles at ˚C for 30 sec, and 72˚C for 25 sec.
Interpretation of the result was performed using the Pfaffle method and the CT values were normalized to the
as a housekeeping gene. All reactions were performed in triplicate
and negative control included in each experiment
One way ANOVA was performed for statistical analysis using SPSS version 18 following with
Genes with corresponding primers which used for gene expression
Accession No.
NM_002046.3
NM_000633.2
NM_005163.2
NM_018947.4
NM_007315.3
NM_001126114
Reverse
Copyright © 2011 by Tabriz University of Medical Sciences
Cells were subjected to gene expression evaluation justobtained as IC50 of CBX
total RNA was isolated using to manufacturer guideline. the extracted RNA was measured
respectively by electrophoresis and NanoDrop®p Technologies,
Primers were designed from sequences using Beacon Designer
(Premier Biosoft International, http://www.premierbiosoft.com) and listed in Table 1.
on reaction was performed using MMLV buffered with
pdN6 (400 ng/RNasin (40 U), and 1
l. Thermal cycler C for 5 min before addition of RNasin
and reverse transcriptase, and incubation at 25 C for 50 min and then they were
real time PCR examinations. PCR experiment was performed to assess the expression of selected key genes
All amplification reactions were performed in a total 25µl volume using the iQ5 Optical System (Bio
nc., Hercules, USA). Each well contained: 1 µl cDNA, 1 µl primer (100 nM each primer), 12.5 µl 2X Power SYBR Green PCR Master Mix and 10.5 µl RNAse/DNAse free water. Thermal cycling conditions
˚C for 10 min, 40 cycles at ˚C for 30 sec, and 72˚C for 25 sec.
Interpretation of the result was performed using the Pfaffle method and the CT values were normalized to the
as a housekeeping gene. All reactions were performed in triplicate
and negative control included in each experiment
One way ANOVA was performed for statistical analysis Tukey’s Post Hoc
Genes with corresponding primers which used for gene expression
Accession No.
NM_002046.3
NM_000633.2
NM_005163.2
NM_018947.4
NM_007315.3
NM_001126114
Copyright © 2011 by Tabriz University of Medical Sciences
Cells were subjected to gene expression evaluation justobtained as IC50 of CBX
total RNA was isolated using to manufacturer guideline. the extracted RNA was measured
respectively by electrophoresis and NanoDrop®p Technologies,
Primers were designed from sequences using Beacon Designer
(Premier Biosoft International, http://www.premierbiosoft.com) and listed in Table 1.
on reaction was performed using MMLV buffered with
pdN6 (400 ng/RNasin (40 U), and 1 l
. Thermal cycler C for 5 min before addition of RNasin
and reverse transcriptase, and incubation at 25 C for 50 min and then they were subjected to
real time PCR examinations. PCR experiment was performed to assess the expression of selected key genes
All amplification reactions were performed in a total 25µl volume using the iQ5 Optical System (Bio
nc., Hercules, USA). Each well contained: 1 µl cDNA, 1 µl primer (100 nM each primer), 12.5 µl 2X Power SYBR Green PCR Master Mix and 10.5 µl RNAse/DNAse free water. Thermal cycling conditions
˚C for 10 min, 40 cycles at ˚C for 30 sec, and 72˚C for 25 sec.
Interpretation of the result was performed using the Pfaffle method and the CT values were normalized to the
as a housekeeping gene. All reactions were performed in triplicate
and negative control included in each experiment
One way ANOVA was performed for statistical analysis Tukey’s Post Hoc
Genes with corresponding primers which used for gene expression
Accession No.
NM_002046.3
NM_000633.2
NM_005163.2
NM_018947.4
NM_007315.3
NM_001126114
Copyright © 2011 by Tabriz University of Medical Sciences
Cells were subjected to gene expression evaluation justobtained as IC50 of CBX
total RNA was isolated using to manufacturer guideline. the extracted RNA was measured
respectively by electrophoresis and NanoDrop®p Technologies,
Primers were designed from sequences using Beacon Designer
(Premier Biosoft International, http://www.premierbiosoft.com) and listed in Table 1.
on reaction was performed using MMLV buffered with
pdN6 (400 ng/l MMLV
. Thermal cycler C for 5 min before addition of RNasin
and reverse transcriptase, and incubation at 25 ˚C for 10 subjected to
real time PCR examinations. PCR experiment was performed to assess the expression of selected key genes
All amplification reactions were performed in a total 25µl volume using the iQ5 Optical System (Bio
nc., Hercules, USA). Each well contained: 1 µl cDNA, 1 µl primer (100 nM each primer), 12.5 µl 2X Power SYBR Green PCR Master Mix and 10.5 µl RNAse/DNAse free water. Thermal cycling conditions
˚C for 10 min, 40 cycles at ˚C for 30 sec, and 72˚C for 25 sec.
Interpretation of the result was performed using the Pfaffle method and the CT values were normalized to the
as a housekeeping gene. All reactions were performed in triplicate
and negative control included in each experiment
One way ANOVA was performed for statistical analysis Tukey’s Post Hoc
Genes with corresponding primers which used for gene expression
Cells were subjected to gene expression evaluation justobtained as IC50 of CBX at the
total RNA was isolated using to manufacturer guideline. the extracted RNA was measured
respectively by electrophoresis and NanoDrop®1000 p Technologies,
Primers were designed from sequences using Beacon Designer
(Premier Biosoft International, http://www.premierbiosoft.com) and listed in Table 1.
on reaction was performed using MMLV buffered with
pdN6 (400 ng/lMMLV
. Thermal cycler C for 5 min before addition of RNasin
C for 10 subjected to
real time PCR examinations. PCR experiment was performed to assess the expression of selected key genes
All amplification reactions were performed in a total 25µl volume using the iQ5 Optical System (Bio-Rad
nc., Hercules, USA). Each well contained: 1 µl cDNA, 1 µl primer (100 nM each primer), 12.5 µl 2X Power SYBR Green PCR Master Mix and 10.5 µl RNAse/DNAse free water. Thermal cycling conditions
˚C for 10 min, 40 cycles at ˚C for 30 sec, and 72˚C for 25 sec.
Interpretation of the result was performed using the Pfaffle method and the CT values were normalized to the
as a housekeeping gene. All reactions were performed in triplicate
as well
One way ANOVA was performed for statistical analysis Tukey’s Post Hoc
Genes with corresponding primers which used for gene expression
Sequence
F: 5’R: 5’F: 5’R: 5’F: 5’R: 5’F: 5’R: 5’F: 5’R: 5’F: 5’R: 5’
Cells were subjected to gene expression evaluation justat the
total RNA was isolated using to manufacturer guideline. The the extracted RNA was measured
1000 p Technologies,
Primers were designed from sequences using Beacon Designer
(Premier Biosoft International, http://www.premierbiosoft.com) and listed in Table 1.
on reaction was performed using MMLV buffered with
l), 1 MMLV-rt
. Thermal cycler C for 5 min before addition of RNasin
C for 10 subjected to
real time PCR examinations. PCR experiment was performed to assess the expression of selected key genes
All amplification reactions were performed in a total Rad
nc., Hercules, USA). Each well contained: 1 µl cDNA, 1 µl primer (100 nM each primer), 12.5 µl 2X Power SYBR Green PCR Master Mix and 10.5 µl RNAse/DNAse free water. Thermal cycling conditions
˚C for 10 min, 40 cycles at ˚C for 30 sec, and 72˚C for 25 sec.
Interpretation of the result was performed using the Pfaffle method and the CT values were normalized to the
as a housekeeping gene. All reactions were performed in triplicate
as well
One way ANOVA was performed for statistical analysis Tukey’s Post Hoc
Genes with corresponding primers which used for gene expression
Sequence
5’-AAGCTCATTTCCTGGTATGACAACGR: 5’-TCTTCCTCTTGTGCTCTTGCTGG
5’-CATCAGGAAGGCTAGAGTTACCR: 5’-CAGACATTCGGAGACCACACF: 5’- CGCAGTGCCAGCTGATR: 5’- GTCCATCTCCTCCTCCTCCTG F: 5’- ACCTTCCATCTTGGCTAGTTGTGR: 5’- ATCGCTTGAGCCTGGGAAATAGF: 5’- TCATCAGCAAGGAGCGAGAGR: 5’- TCAGGGAAAGTAACAGCAGAAAGF: 5’-TCAACAAGATGTTTTGCCAACTGR: 5’-ATGTGCTGTGA
Cells were subjected to gene expression evaluation just at the
total RNA was isolated using The
the extracted RNA was measured 1000
p Technologies, Primers were designed from
sequences using Beacon Designer (Premier Biosoft International,
http://www.premierbiosoft.com) and listed in Table 1. on reaction was performed using
MMLV buffered with ), 1
rt . Thermal cycler
C for 5 min before addition of RNasin C for 10
subjected to real time PCR examinations. PCR experiment was performed to assess the expression of selected key genes
All amplification reactions were performed in a total Rad
nc., Hercules, USA). Each well contained: 1 µl cDNA, 1 µl primer (100 nM each primer), 12.5 µl 2X Power SYBR Green PCR Master Mix and 10.5 µl RNAse/DNAse free water. Thermal cycling conditions
˚C for 10 min, 40 cycles at ˚C for 30 sec, and 72˚C for 25 sec.
Interpretation of the result was performed using the Pfaffle method and the CT values were normalized to the
as a housekeeping gene . All reactions were performed in triplicate
as well
One way ANOVA was performed for statistical analysis Tukey’s Post Hoc
Genes with corresponding primers which used for gene expression
Sequence
AAGCTCATTTCCTGGTATGACAACGTCTTCCTCTTGTGCTCTTGCTGGCATCAGGAAGGCTAGAGTTACCCAGACATTCGGAGACCACACCGCAGTGCCAGCTGATGTCCATCTCCTCCTCCTCCTG ACCTTCCATCTTGGCTAGTTGTGATCGCTTGAGCCTGGGAAATAGTCATCAGCAAGGAGCGAGAGTCAGGGAAAGTAACAGCAGAAAG
TCAACAAGATGTTTTGCCAACTGATGTGCTGTGA
Genes with corresponding primers which used for gene expression
AAGCTCATTTCCTGGTATGACAACGTCTTCCTCTTGTGCTCTTGCTGGCATCAGGAAGGCTAGAGTTACCCAGACATTCGGAGACCACACCGCAGTGCCAGCTGATGTCCATCTCCTCCTCCTCCTG ACCTTCCATCTTGGCTAGTTGTGATCGCTTGAGCCTGGGAAATAGTCATCAGCAAGGAGCGAGAGTCAGGGAAAGTAACAGCAGAAAG
TCAACAAGATGTTTTGCCAACTGATGTGCTGTGA
Teststatistical differences representation.Trypan blue exclusion testsfive independent evaluationtriplicate
Results
Cytotoxicity assessmentTrypan blue exclusion test and MTT assay were conducted to evaluate the cytotoxicity of carbenoxolone in MCF7and BT20 human breast cancer cells. As shown in Fig.dose dependent manner
Fig. BT20 cells(50well as for 24 h (number of viable cellstest. Growth inhibition in each treatment was expressed as a percentage of the control. Each value represents the meanof at least five
Genes with corresponding primers which used for gene expression
AAGCTCATTTCCTGGTATGACAACGTCTTCCTCTTGTGCTCTTGCTGGCATCAGGAAGGCTAGAGTTACCCAGACATTCGGAGACCACACCGCAGTGCCAGCTGATGTCCATCTCCTCCTCCTCCTG ACCTTCCATCTTGGCTAGTTGTGATCGCTTGAGCCTGGGAAATAGTCATCAGCAAGGAGCGAGAGTCAGGGAAAGTAACAGCAGAAAG
TCAACAAGATGTTTTGCCAACTGATGTGCTGTGACTGCTTGTAGATG
Test. A statistical differences representation.Trypan blue exclusion testsfive independent evaluationtriplicate
Results
Cytotoxicity assessmentTrypan blue exclusion test and MTT assay were conducted to evaluate the cytotoxicity of carbenoxolone in MCF7and BT20 human breast cancer cells. As shown in Fig.dose dependent manner
Fig. 2BT20 cells(50-1000µM) for 24 h (well as for 24 h (number of viable cellstest. Growth inhibition in each treatment was expressed as a percentage of the control. Each value represents the meanof at least five
Genes with corresponding primers which used for gene expression
AAGCTCATTTCCTGGTATGACAACGTCTTCCTCTTGTGCTCTTGCTGGCATCAGGAAGGCTAGAGTTACCCAGACATTCGGAGACCACACCGCAGTGCCAGCTGATGTCCATCTCCTCCTCCTCCTG ACCTTCCATCTTGGCTAGTTGTGATCGCTTGAGCCTGGGAAATAGTCATCAGCAAGGAGCGAGAGTCAGGGAAAGTAACAGCAGAAAG
TCAACAAGATGTTTTGCCAACTGCTGCTTGTAGATG
. A statistical differences representation.Trypan blue exclusion testsfive independent evaluationtriplicate
Results
Cytotoxicity assessmentTrypan blue exclusion test and MTT assay were conducted to evaluate the cytotoxicity of carbenoxolone in MCF7and BT20 human breast cancer cells. As shown in Fig. 2dose dependent manner
2. The BT20 cells
1000µM) for 24 h (well as for 24 h (number of viable cellstest. Growth inhibition in each treatment was expressed as a percentage of the control. Each value represents the meanof at least five
Genes with corresponding primers which used for gene expression
AAGCTCATTTCCTGGTATGACAACGTCTTCCTCTTGTGCTCTTGCTGGCATCAGGAAGGCTAGAGTTACCCAGACATTCGGAGACCACACCGCAGTGCCAGCTGATGAAG GTCCATCTCCTCCTCCTCCTG ACCTTCCATCTTGGCTAGTTGTGATCGCTTGAGCCTGGGAAATAGTCATCAGCAAGGAGCGAGAGTCAGGGAAAGTAACAGCAGAAAG
TCAACAAGATGTTTTGCCAACTGCTGCTTGTAGATG
. A pstatistical differences representation.Trypan blue exclusion testsfive independent evaluation triplicate manner.
Cytotoxicity assessmentTrypan blue exclusion test and MTT assay were conducted to evaluate the cytotoxicity of carbenoxolone in MCF7and BT20 human breast cancer cells. As shown
2, CBX treated cells led to dose dependent manner
The Effects of CBX on growth inhibition of MCF7 and BT20 cells. Cells were exposed to the indicated concentration
1000µM) for 24 h (well as for 24 h (number of viable cellstest. Growth inhibition in each treatment was expressed as a percentage of the control. Each value represents the meanof at least five
Genes with corresponding primers which used for gene expression
AAGCTCATTTCCTGGTATGACAACGTCTTCCTCTTGTGCTCTTGCTGGCATCAGGAAGGCTAGAGTTACCCAGACATTCGGAGACCACAC-3’
GAAG GTCCATCTCCTCCTCCTCCTG -ACCTTCCATCTTGGCTAGTTGTGATCGCTTGAGCCTGGGAAATAGTCATCAGCAAGGAGCGAGAG-TCAGGGAAAGTAACAGCAGAAAG
TCAACAAGATGTTTTGCCAACTGCTGCTTGTAGATG
p valuestatistical differences representation.Trypan blue exclusion testsfive independent
using real time RTmanner.
Cytotoxicity assessmentTrypan blue exclusion test and MTT assay were conducted to evaluate the cytotoxicity of carbenoxolone in MCF7and BT20 human breast cancer cells. As shown
, CBX treated cells led to dose dependent manner
Effects of CBX on growth inhibition of MCF7 and Cells were exposed to the indicated concentration
1000µM) for 24 h (well as for 24 h (number of viable cellstest. Growth inhibition in each treatment was expressed as a percentage of the control. Each value represents the meanof at least five independent experiments
Genes with corresponding primers which used for gene expression
AAGCTCATTTCCTGGTATGACAACGTCTTCCTCTTGTGCTCTTGCTGG-3’ CATCAGGAAGGCTAGAGTTACC-3’
3’ GAAG -3’
-3’ ACCTTCCATCTTGGCTAGTTGTG-3’ATCGCTTGAGCCTGGGAAATAG-3’
-3’ TCAGGGAAAGTAACAGCAGAAAG
TCAACAAGATGTTTTGCCAACTG-3’CTGCTTGTAGATG-
valuestatistical differences representation.Trypan blue exclusion testsfive independent
using real time RTmanner.
Cytotoxicity assessmentTrypan blue exclusion test and MTT assay were conducted to evaluate the cytotoxicity of carbenoxolone in MCF7and BT20 human breast cancer cells. As shown
, CBX treated cells led to dose dependent manner
Effects of CBX on growth inhibition of MCF7 and Cells were exposed to the indicated concentration
1000µM) for 24 h (well as for 24 h (▲) and 48 number of viable cellstest. Growth inhibition in each treatment was expressed as a percentage of the control. Each value represents the mean
independent experiments
Genes with corresponding primers which used for gene expression
AAGCTCATTTCCTGGTATGACAACG-3’
3’ 3’
TCAGGGAAAGTAACAGCAGAAAG-3’ 3’
-3’
BioImpacts
value less than 0.05 was considered to statistical differences representation.Trypan blue exclusion testsfive independent experiments
using real time RT
Cytotoxicity assessmentTrypan blue exclusion test and MTT assay were conducted to evaluate the cytotoxicity of carbenoxolone in MCF7and BT20 human breast cancer cells. As shown
, CBX treated cells led to dose dependent manner
Effects of CBX on growth inhibition of MCF7 and Cells were exposed to the indicated concentration
1000µM) for 24 h (♦) and 48 ▲) and 48
number of viable cells was determined by Trypan blue exclusion test. Growth inhibition in each treatment was expressed as a percentage of the control. Each value represents the mean
independent experiments
Genes with corresponding primers which used for gene expression
ioImpacts
less than 0.05 was considered to statistical differences representation.Trypan blue exclusion tests
experimentsusing real time RT
Cytotoxicity assessment Trypan blue exclusion test and MTT assay were conducted to evaluate the cytotoxicity of carbenoxolone in MCF7and BT20 human breast cancer cells. As shown
, CBX treated cells led to dose dependent manner (p < 0.05)
Effects of CBX on growth inhibition of MCF7 and Cells were exposed to the indicated concentration
♦) and 48 ▲) and 48
was determined by Trypan blue exclusion test. Growth inhibition in each treatment was expressed as a percentage of the control. Each value represents the mean
independent experiments
Genes with corresponding primers which used for gene expression analysis
Product length
ioImpacts
less than 0.05 was considered to statistical differences representation.Trypan blue exclusion tests were performed in
experimentsusing real time RT
Trypan blue exclusion test and MTT assay were conducted to evaluate the cytotoxicity of carbenoxolone in MCF7and BT20 human breast cancer cells. As shown
, CBX treated cells led to (p < 0.05)
Effects of CBX on growth inhibition of MCF7 and Cells were exposed to the indicated concentration
♦) and 48 ▲) and 48 h (
was determined by Trypan blue exclusion test. Growth inhibition in each treatment was expressed as a percentage of the control. Each value represents the mean
independent experiments
analysis
Product length
126
181
185
129
196
118
ioImpacts, 2011, 1(2),
less than 0.05 was considered to statistical differences representation.
were performed in experiments
using real time RT
Trypan blue exclusion test and MTT assay were conducted to evaluate the cytotoxicity of carbenoxolone in MCF7and BT20 human breast cancer cells. As shown
, CBX treated cells led to (p < 0.05)
Effects of CBX on growth inhibition of MCF7 and Cells were exposed to the indicated concentration
♦) and 48 h (□) in the case of MCF7 as h (■) in the case of BT20. The
was determined by Trypan blue exclusion test. Growth inhibition in each treatment was expressed as a percentage of the control. Each value represents the mean
independent experiments
analysis
Product length
126
181
185
129
196
118
, 2011, 1(2),
less than 0.05 was considered to statistical differences representation.
were performed in and gene expression
using real time RT-PCR performed
Trypan blue exclusion test and MTT assay were conducted to evaluate the cytotoxicity of carbenoxolone in MCF7and BT20 human breast cancer cells. As shown
, CBX treated cells led to growth inhibition in (p < 0.05).
Effects of CBX on growth inhibition of MCF7 and Cells were exposed to the indicated concentration
□) in the case of MCF7 as ■) in the case of BT20. The
was determined by Trypan blue exclusion test. Growth inhibition in each treatment was expressed as a percentage of the control. Each value represents the mean
independent experiments.
Product length
, 2011, 1(2),
less than 0.05 was considered to statistical differences representation. MTT assays a
were performed in and gene expression
PCR performed
Trypan blue exclusion test and MTT assay were conducted to evaluate the cytotoxicity of carbenoxolone in MCF7and BT20 human breast cancer cells. As shown
growth inhibition in
Effects of CBX on growth inhibition of MCF7 and Cells were exposed to the indicated concentration
□) in the case of MCF7 as ■) in the case of BT20. The
was determined by Trypan blue exclusion test. Growth inhibition in each treatment was expressed as a percentage of the control. Each value represents the mean
Product length
, 2011, 1(2), 113-
less than 0.05 was considered to MTT assays a
were performed in and gene expression
PCR performed
Trypan blue exclusion test and MTT assay were conducted to evaluate the cytotoxicity of carbenoxolone in MCF7and BT20 human breast cancer cells. As shown
growth inhibition in
Effects of CBX on growth inhibition of MCF7 and Cells were exposed to the indicated concentration
□) in the case of MCF7 as ■) in the case of BT20. The
was determined by Trypan blue exclusion test. Growth inhibition in each treatment was expressed as a percentage of the control. Each value represents the mean
-119
less than 0.05 was considered to MTT assays a
were performed in and gene expression
PCR performed
Trypan blue exclusion test and MTT assay were conducted to evaluate the cytotoxicity of carbenoxolone in MCF7and BT20 human breast cancer cells. As shown
growth inhibition in
Effects of CBX on growth inhibition of MCF7 and Cells were exposed to the indicated concentration
□) in the case of MCF7 as ■) in the case of BT20. The
was determined by Trypan blue exclusion test. Growth inhibition in each treatment was expressed as a percentage of the control. Each value represents the mean
less than 0.05 was considered to MTT assays a
were performed in at least and gene expression
PCR performed
Trypan blue exclusion test and MTT assay were conducted to evaluate the cytotoxicity of carbenoxolone in MCF7and BT20 human breast cancer cells. As shown
growth inhibition in
Effects of CBX on growth inhibition of MCF7 and Cells were exposed to the indicated concentration
□) in the case of MCF7 as ■) in the case of BT20. The
was determined by Trypan blue exclusion test. Growth inhibition in each treatment was expressed as a percentage of the control. Each value represents the mean
| 115
less than 0.05 was considered to MTT assays a
at least and gene expression
PCR performed
Trypan blue exclusion test and MTT assay were conducted to evaluate the cytotoxicity of carbenoxolone in MCF7and BT20 human breast cancer cells. As shown
growth inhibition in
Effects of CBX on growth inhibition of MCF7 and Cells were exposed to the indicated concentration
□) in the case of MCF7 as ■) in the case of BT20. The
was determined by Trypan blue exclusion test. Growth inhibition in each treatment was expressed as a percentage of the control. Each value represents the mean ± SE
115
less than 0.05 was considered to MTT assays and
at least and gene expression
in
Trypan blue exclusion test and MTT assay were conducted to evaluate the cytotoxicity of carbenoxolone in MCF7and BT20 human breast cancer cells. As shown
growth inhibition in
Effects of CBX on growth inhibition of MCF7 and Cells were exposed to the indicated concentration
□) in the case of MCF7 as ■) in the case of BT20. The
was determined by Trypan blue exclusion test. Growth inhibition in each treatment was expressed as a
SE
Asadi-Khiavi et al.
116 | BioImpacts, 2011, 1(2), 113-119 Copyright © 2011 by Tabriz University of Medical Sciences
Fig. 3. Photomicrographs of MCF7 and BT20 cells were taken by a light microscope at a magnification of 40×. MCF7 and BT20 cells were treated by 150 µM of CBX. The death cells are shown by black arrows. Panel A represent untreated MCF7 cells versus CBX treated MCF7 cells in panels B and C at exposure times of 24h and 48h respectively. Panel D represent untreated BT20 cells versus CBX treated BT20 cells in panels E and F at exposure times of 24h and 48h respectively.
As it shows, the growth of treated BT20 cells was affected notably, but in the case of MCF7 cells, anti-proliferative effect of CBX was lower than previous cells. However, CBX also showed statistically significant cytotoxicity in the MCF cells. Additionally, the treated and untreated cells displayed distinct morphologic differences in normal and death cells number and appearance upon light microscopic examinations (Fig. 3). Regarding the obtained data from MTT experiment as a standard colorimetric cell viability assay, IC50 were calculated as 150 M in both
cells (Fig 4), which is relatively similar to obtained IC50 from data of growth inhibition evaluation via Trypan blue exclusion test in Fig. 2. In the view of statistics (mean ±SE of at least five independent experiments), reduction in number of cells was observed after treatment by 150 µM of CBX as same as reduction in total amount of extracted RNA in both cells (data not shown). Despite mild exacerbation in CBX mediated cytotoxicity in a time dependent manner, it was not statistically significant even at exposure time of 72 hr in both cell lines (data not shown).
A
B
C
D
E
F
MCF7 Cells BT20 Cells
24h 24h
48h 48h
Untreated cells Untreated cells
Gap junctions in cancer cells
Copyright © 2011 by Tabriz University of Medical Sciences
Fig. were exposed to 1000µM) at exposure time of 24 h (repeated in the case of BT20 for mentioned concentrations and exposure timesconcentration could significantlyformazan as reductive metabolite of MTT (3Dimethylthiazolcell linesindependent experiments.
Gene expression analyses MCF7 and BT20 cells were incubµM) for 24 h. Twhich mentioned above were detected by using real time PCR andusing the Pfafeach geneGAPDHperformed in triplicate and negative control included in each experimentGreen
We effect related genesgenesthe cell cycle is involved in cancer1999GAPDHrespective ratio of expressed gene overexpression was used to represent gene expression changesNormalization of our data to the expression rate of GAPDHdown regulationcells but candidate genes
Gap junctions in cancer cells
Copyright © 2011 by Tabriz University of Medical Sciences
Fig. 4. Rwere exposed to 1000µM) at exposure time of 24 h (repeated in the case of BT20 for mentioned concentrations and exposure timesconcentration could significantlyformazan as reductive metabolite of MTT (3Dimethylthiazolcell linesindependent experiments.
Gene expression analyses MCF7 and BT20 cells were incubµM) for 24 h. Twhich mentioned above were detected by using real time PCR andusing the Pfafeach geneGAPDHperformed in triplicate and negative control included in each experimentGreen emitted fluorescent
We performedeffect of carbenoxolone related genesgenes AKT1the cell cycle is involved in cancer1999). GAPDHrespective ratio of expressed gene overexpression was used to represent gene expression changesNormalization of our data to the expression rate of GAPDHdown regulationcells but candidate genes
Gap junctions in cancer cells
Copyright © 2011 by Tabriz University of Medical Sciences
Results of MTT assay for evaluation were exposed to 1000µM) at exposure time of 24 h (repeated in the case of BT20 for mentioned concentrations and exposure timesconcentration could significantlyformazan as reductive metabolite of MTT (3Dimethylthiazolcell lines. Each value represents the mean ±independent experiments.
Gene expression analyses MCF7 and BT20 cells were incubµM) for 24 h. Twhich mentioned above were detected by using real time PCR and using the Pfafeach gene GAPDH as a housekeeping geneperformed in triplicate and negative control included in each experiment
emitted fluorescent
performedof carbenoxolone
related genesAKT1
the cell cycle is involved in cancer
. The GAPDH expression isrespective ratio of expressed gene overexpression was used to represent gene expression changes and was demonstratedNormalization of our data to the expression rate of GAPDH as a housekeeping down regulationcells but there was not candidate genes
Gap junctions in cancer cells
Copyright © 2011 by Tabriz University of Medical Sciences
esults of MTT assay for evaluation were exposed to 1000µM) at exposure time of 24 h (repeated in the case of BT20 for mentioned concentrations and exposure times.concentration could significantlyformazan as reductive metabolite of MTT (3Dimethylthiazol-
Each value represents the mean ±independent experiments.
Gene expression analyses MCF7 and BT20 cells were incubµM) for 24 h. Twhich mentioned above were detected by using real time
Interpretation of the resultusing the Pfaf
were normalized to the expressionas a housekeeping gene
performed in triplicate and negative control included in each experiment
emitted fluorescent
performedof carbenoxolone
related genes BCL2AKT1and
the cell cycle control is involved in cancer
The mRNA expression is
respective ratio of expressed gene overexpression was used to represent gene expression
and was demonstratedNormalization of our data to the expression rate of
as a housekeeping down regulation
there was not candidate genes
Gap junctions in cancer cells
Copyright © 2011 by Tabriz University of Medical Sciences
esults of MTT assay for evaluation were exposed to 1000µM) at exposure time of 24 h (repeated in the case of BT20 for mentioned concentrations and
. As shown in concentration could significantlyformazan as reductive metabolite of MTT (3
-2-yl)Each value represents the mean ±
independent experiments.
Gene expression analyses MCF7 and BT20 cells were incubµM) for 24 h. Thewhich mentioned above were detected by using real time
Interpretation of the resultusing the Pfaffle method and
were normalized to the expressionas a housekeeping gene
performed in triplicate and negative control included in each experiment and then quantified as a view of
emitted fluorescent
performed a quantitative PCR approach to assess the of carbenoxolone
BCL2and STAT5
control is involved in cancer
mRNA expression is
respective ratio of expressed gene overexpression was used to represent gene expression
and was demonstratedNormalization of our data to the expression rate of
as a housekeeping down regulation of
there was not candidate genes themselves
Gap junctions in cancer cells
Copyright © 2011 by Tabriz University of Medical Sciences
esults of MTT assay for evaluation were exposed to the indicated concentration of CBX (501000µM) at exposure time of 24 h (repeated in the case of BT20 for mentioned concentrations and
As shown in concentration could significantlyformazan as reductive metabolite of MTT (3
yl)-2, 5Each value represents the mean ±
independent experiments.
Gene expression analyses MCF7 and BT20 cells were incub
hen expression levels of candidate genes which mentioned above were detected by using real time
Interpretation of the resultfle method and
were normalized to the expressionas a housekeeping gene
performed in triplicate and negative control included in and then quantified as a view of
emitted fluorescent
a quantitative PCR approach to assess the of carbenoxolone
BCL2 andSTAT5
control is involved in cancer
mRNA expression is
respective ratio of expressed gene overexpression was used to represent gene expression
and was demonstratedNormalization of our data to the expression rate of
as a housekeeping of all
there was not themselves
Gap junctions in cancer cells
Copyright © 2011 by Tabriz University of Medical Sciences
esults of MTT assay for evaluation the indicated concentration of CBX (50
1000µM) at exposure time of 24 h (repeated in the case of BT20 for mentioned concentrations and
As shown in concentration could significantlyformazan as reductive metabolite of MTT (3
, 5-diphenyltetrazolium bromide) in Each value represents the mean ±
independent experiments.
Gene expression analyses MCF7 and BT20 cells were incub
expression levels of candidate genes which mentioned above were detected by using real time
Interpretation of the resultfle method and
were normalized to the expressionas a housekeeping gene
performed in triplicate and negative control included in and then quantified as a view of
emitted fluorescent
a quantitative PCR approach to assess the of carbenoxolone
and STAT5and finally
control and a tumor suppressoris involved in cancer preventing
mRNA expression of these genes versus expression is
respective ratio of expressed gene overexpression was used to represent gene expression
and was demonstratedNormalization of our data to the expression rate of
as a housekeeping all mentioned
there was not significant changes among these themselves
Copyright © 2011 by Tabriz University of Medical Sciences
esults of MTT assay for evaluation the indicated concentration of CBX (50
1000µM) at exposure time of 24 h (repeated in the case of BT20 for mentioned concentrations and
As shown in this diagramconcentration could significantlyformazan as reductive metabolite of MTT (3
diphenyltetrazolium bromide) in Each value represents the mean ±
Gene expression analyses MCF7 and BT20 cells were incub
expression levels of candidate genes which mentioned above were detected by using real time
Interpretation of the resultfle method and
were normalized to the expressionas a housekeeping gene
performed in triplicate and negative control included in and then quantified as a view of
emitted fluorescent intensity.
a quantitative PCR approach to assess the of carbenoxolone on the expression
CYSCand finally
and a tumor suppressorpreventing
expression of these genes versus illustrated in Figure 5. The
respective ratio of expressed gene overexpression was used to represent gene expression
and was demonstratedNormalization of our data to the expression rate of
as a housekeeping mentioned significant changes among these
themselves.
Copyright © 2011 by Tabriz University of Medical Sciences
esults of MTT assay for evaluation the indicated concentration of CBX (50
1000µM) at exposure time of 24 h (♦) and 48 h (□) and also arerepeated in the case of BT20 for mentioned concentrations and
this diagramconcentration could significantly suppress the production of formazan as reductive metabolite of MTT (3
diphenyltetrazolium bromide) in Each value represents the mean ±
Gene expression analyses MCF7 and BT20 cells were incub
expression levels of candidate genes which mentioned above were detected by using real time
Interpretation of the resultfle method and finally
were normalized to the expressionas a housekeeping gene
performed in triplicate and negative control included in and then quantified as a view of
intensity.
a quantitative PCR approach to assess the on the expression CYSC ,
and finallyand a tumor suppressorpreventing
expression of these genes versus illustrated in Figure 5. The
respective ratio of expressed gene overexpression was used to represent gene expression
and was demonstrated Normalization of our data to the expression rate of
as a housekeeping gene, mentioned significant changes among these
Copyright © 2011 by Tabriz University of Medical Sciences
esults of MTT assay for evaluation the indicated concentration of CBX (50
♦) and 48 h (□) and also arerepeated in the case of BT20 for mentioned concentrations and
this diagramsuppress the production of
formazan as reductive metabolite of MTT (3diphenyltetrazolium bromide) in
Each value represents the mean ±
MCF7 and BT20 cells were incubexpression levels of candidate genes
which mentioned above were detected by using real time Interpretation of the result
finallywere normalized to the expression
as a housekeeping geneperformed in triplicate and negative control included in
and then quantified as a view ofintensity.
a quantitative PCR approach to assess the on the expression
, EGF pathway candidate and finally
and a tumor suppressorpreventing ( expression of these genes versus
illustrated in Figure 5. The respective ratio of expressed gene overexpression was used to represent gene expression
quantitatively Normalization of our data to the expression rate of
gene, mentioned genes in both type of significant changes among these
Copyright © 2011 by Tabriz University of Medical Sciences
esults of MTT assay for evaluation the indicated concentration of CBX (50
♦) and 48 h (□) and also arerepeated in the case of BT20 for mentioned concentrations and
this diagram, CBX in 150 suppress the production of
formazan as reductive metabolite of MTT (3diphenyltetrazolium bromide) in
Each value represents the mean ±
MCF7 and BT20 cells were incubated with CBX (150 expression levels of candidate genes
which mentioned above were detected by using real time Interpretation of the result
finally were normalized to the expression
as a housekeeping gene. All reactions were performed in triplicate and negative control included in
and then quantified as a view ofintensity.
a quantitative PCR approach to assess the on the expression
EGF pathway candidate TP53
and a tumor suppressor May, P. and May, E.
expression of these genes versus illustrated in Figure 5. The
respective ratio of expressed gene overexpression was used to represent gene expression
quantitatively Normalization of our data to the expression rate of
gene, revealed significantgenes in both type of
significant changes among these
Copyright © 2011 by Tabriz University of Medical Sciences
esults of MTT assay for evaluation of cell viability. the indicated concentration of CBX (50
♦) and 48 h (□) and also arerepeated in the case of BT20 for mentioned concentrations and
, CBX in 150 suppress the production of
formazan as reductive metabolite of MTT (3diphenyltetrazolium bromide) in
Each value represents the mean ± SE of at least five
ated with CBX (150 expression levels of candidate genes
which mentioned above were detected by using real time Interpretation of the results was performed
the CT valueswere normalized to the expression
. All reactions were performed in triplicate and negative control included in
and then quantified as a view of
a quantitative PCR approach to assess the on the expression
EGF pathway candidate TP53 as
and a tumor suppressorMay, P. and May, E.
expression of these genes versus illustrated in Figure 5. The
respective ratio of expressed gene overexpression was used to represent gene expression
quantitatively Normalization of our data to the expression rate of
revealed significantgenes in both type of
significant changes among these
Copyright © 2011 by Tabriz University of Medical Sciences
of cell viability. the indicated concentration of CBX (50
♦) and 48 h (□) and also arerepeated in the case of BT20 for mentioned concentrations and
, CBX in 150 suppress the production of
formazan as reductive metabolite of MTT (3diphenyltetrazolium bromide) in
SE of at least five
ated with CBX (150 expression levels of candidate genes
which mentioned above were detected by using real time was performed
the CT valueswere normalized to the expression behavior
. All reactions were performed in triplicate and negative control included in
and then quantified as a view of
a quantitative PCR approach to assess the on the expression of
EGF pathway candidate as crucial
and a tumor suppressor geneMay, P. and May, E.
expression of these genes versus illustrated in Figure 5. The
respective ratio of expressed gene overexpression was used to represent gene expression
quantitatively Normalization of our data to the expression rate of
revealed significantgenes in both type of
significant changes among these
Copyright © 2011 by Tabriz University of Medical Sciences
of cell viability. the indicated concentration of CBX (50
♦) and 48 h (□) and also arerepeated in the case of BT20 for mentioned concentrations and
, CBX in 150 suppress the production of
formazan as reductive metabolite of MTT (3diphenyltetrazolium bromide) in
SE of at least five
ated with CBX (150 expression levels of candidate genes
which mentioned above were detected by using real time was performed
the CT valuesbehavior
. All reactions were performed in triplicate and negative control included in
and then quantified as a view of
a quantitative PCR approach to assess the of apoptosis
EGF pathway candidate crucialgene
May, P. and May, E. expression of these genes versus
illustrated in Figure 5. The respective ratio of expressed gene over GAPDHexpression was used to represent gene expression
quantitatively in Fig. 5Normalization of our data to the expression rate of
revealed significantgenes in both type of
significant changes among these
of cell viability. Cthe indicated concentration of CBX (50
♦) and 48 h (□) and also arerepeated in the case of BT20 for mentioned concentrations and
, CBX in 150 µM of suppress the production of
formazan as reductive metabolite of MTT (3-(4, 5diphenyltetrazolium bromide) in
SE of at least five
ated with CBX (150 expression levels of candidate genes
which mentioned above were detected by using real time was performed
the CT valuesbehavior
. All reactions were performed in triplicate and negative control included in
and then quantified as a view of SYBR
a quantitative PCR approach to assess the apoptosis
EGF pathway candidate crucial genegene which
May, P. and May, E. expression of these genes versus
illustrated in Figure 5. The GAPDH
expression was used to represent gene expression in Fig. 5
Normalization of our data to the expression rate of revealed significant
genes in both type of significant changes among these
Cells
the indicated concentration of CBX (50-♦) and 48 h (□) and also are
repeated in the case of BT20 for mentioned concentrations and M of
suppress the production of , 5-
diphenyltetrazolium bromide) in both SE of at least five
ated with CBX (150 expression levels of candidate genes
which mentioned above were detected by using real time was performed
the CT values of behavior of
. All reactions were performed in triplicate and negative control included in
SYBR
a quantitative PCR approach to assess the apoptosis
EGF pathway candidate gene
whichMay, P. and May, E.
expression of these genes versus illustrated in Figure 5. The
GAPDHexpression was used to represent gene expression
in Fig. 5. Normalization of our data to the expression rate of
revealed significantgenes in both type of
significant changes among these
ells -
♦) and 48 h (□) and also are repeated in the case of BT20 for mentioned concentrations and
M of suppress the production of
-both
SE of at least five
ated with CBX (150 expression levels of candidate genes
which mentioned above were detected by using real time was performed
of of
. All reactions were performed in triplicate and negative control included in
SYBR
a quantitative PCR approach to assess the apoptosis
EGF pathway candidate gene
which May, P. and May, E.
expression of these genes versus illustrated in Figure 5. The
GAPDH expression was used to represent gene expression
. Normalization of our data to the expression rate of
revealed significant genes in both type of
significant changes among these
Fig. MCF7relative expressions intensity ratio of the genes upon the expression of housekeeping expressions arewere intensity in triplicate experiments statistically (mean ±
DiscussionAlthough, various drugs have been proposed for the treatment of breast cancer, cancer biology andanticancer drugs are obstacles of complete Consequently, many efforts are in progress to find new approaches to overcome cancer.junctions is one of these efforts and CBX is a prototypic inhibitor of them.amount of both typetheIt should be highlighted that MCF7 cells have more proliferative and progressive behaviortheirassay revealed significant reduction in cell viability BT20 cellseffectscells
Fig. 5MCF7relative expressions intensity ratio of the genes upon the expression of housekeeping expressions arewere intensity in triplicate experiments statistically (mean ±
DiscussionAlthough, various drugs have been proposed for the treatment of breast cancer, cancer biology andanticancer drugs are obstacles of complete Consequently, many efforts are in progress to find new approaches to overcome cancer.junctions is one of these efforts and CBX is a prototypic inhibitor of them.amount of both typethe reductionIt should be highlighted that MCF7 cells have more proliferative and progressive behaviortheir assay revealed significant reduction in cell viability BT20 cellseffectscells
Ratio
of G
ene
Expr
essio
n Ra
tio o
f Gen
e Ex
pres
sion
5. Quantitative real time PCR MCF7 cells (Panel A) and BT20 cells relative expressions intensity ratio of the genes upon the expression of housekeeping expressions arewere obtained as a intensity in triplicate experiments statistically (mean ±
DiscussionAlthough, various drugs have been proposed for the treatment of breast cancer, cancer biology andanticancer drugs are obstacles of complete Consequently, many efforts are in progress to find new approaches to overcome cancer.junctions is one of these efforts and CBX is a prototypic inhibitor of them.amount of both type
reductionIt should be highlighted that MCF7 cells have more proliferative and progressive behavior
EGF, progesterone and estrogen receptors.assay revealed significant reduction in cell viability BT20 cellseffects due to mentioned supportive receptors in MCF7 cells (Rulli, Antognelli
0.10.20.30.40.50.60.70.8
Ratio
of G
ene
Expr
essio
n 0.10.20.30.40.50.60.7
Ratio
of G
ene
Expr
essio
n
Quantitative real time PCR cells (Panel A) and BT20 cells
relative expressions intensity ratio of the genes upon the expression of housekeeping expressions are
obtained as a intensity in triplicate experiments statistically (mean ±
Discussion Although, various drugs have been proposed for the treatment of breast cancer, cancer biology andanticancer drugs are obstacles of complete Consequently, many efforts are in progress to find new approaches to overcome cancer.junctions is one of these efforts and CBX is a prototypic inhibitor of them.amount of extracted both types of cells (data not shown) which could indicate
reductionIt should be highlighted that MCF7 cells have more proliferative and progressive behavior
EGF, progesterone and estrogen receptors.assay revealed significant reduction in cell viability BT20 cells
due to mentioned supportive receptors in MCF7 (Rulli, Antognelli
00.10.20.30.40.50.60.70.8
00.10.20.30.40.50.60.7
Quantitative real time PCR cells (Panel A) and BT20 cells
relative expressions intensity ratio of the genes upon the expression of housekeeping expressions are significant statistically
obtained as a intensity in triplicate experiments statistically (mean ±
Although, various drugs have been proposed for the treatment of breast cancer, cancer biology andanticancer drugs are obstacles of complete Consequently, many efforts are in progress to find new approaches to overcome cancer.junctions is one of these efforts and CBX is a prototypic inhibitor of them.
extracted of cells (data not shown) which could indicate
reduction of cell numbersIt should be highlighted that MCF7 cells have more proliferative and progressive behavior
EGF, progesterone and estrogen receptors.assay revealed significant reduction in cell viability
(Fig. 4), while MCF7 did not show due to mentioned supportive receptors in MCF7
(Rulli, Antognelli
TP
TP
Quantitative real time PCR cells (Panel A) and BT20 cells
relative expressions intensity ratio of the genes upon the expression of housekeeping
significant statisticallyobtained as a
intensity in triplicate experiments statistically (mean ±
Although, various drugs have been proposed for the treatment of breast cancer, cancer biology andanticancer drugs are obstacles of complete Consequently, many efforts are in progress to find new approaches to overcome cancer.junctions is one of these efforts and CBX is a prototypic inhibitor of them.
extracted of cells (data not shown) which could indicate
of cell numbersIt should be highlighted that MCF7 cells have more proliferative and progressive behavior
EGF, progesterone and estrogen receptors.assay revealed significant reduction in cell viability
(Fig. 4), while MCF7 did not show due to mentioned supportive receptors in MCF7
(Rulli, Antognelli
TP53
TP53
BioImpacts
Quantitative real time PCR cells (Panel A) and BT20 cells
relative expressions intensity ratio of the genes upon the expression of housekeeping
significant statisticallyobtained as a view of
intensity in triplicate experiments statistically (mean ±
Although, various drugs have been proposed for the treatment of breast cancer, cancer biology and anticancer drugs are obstacles of complete Consequently, many efforts are in progress to find new approaches to overcome cancer.junctions is one of these efforts and CBX is a prototypic
However, in the case of CBX,extracted RNA decreased in treated group in of cells (data not shown) which could indicate
of cell numbersIt should be highlighted that MCF7 cells have more proliferative and progressive behavior
EGF, progesterone and estrogen receptors.assay revealed significant reduction in cell viability
(Fig. 4), while MCF7 did not show due to mentioned supportive receptors in MCF7
(Rulli, Antognelli
53 STAT
MCF
53 STAT
BT
ioImpacts
Quantitative real time PCR cells (Panel A) and BT20 cells
relative expressions intensity ratio of the genes upon the expression of housekeeping
significant statisticallyview of
intensity in triplicate experiments statistically (mean ±
Although, various drugs have been proposed for the treatment of breast cancer,
adverse side effects of routine anticancer drugs are obstacles of complete Consequently, many efforts are in progress to find new approaches to overcome cancer.junctions is one of these efforts and CBX is a prototypic
However, in the case of CBX,RNA decreased in treated group in
of cells (data not shown) which could indicate of cell numbers
It should be highlighted that MCF7 cells have more proliferative and progressive behavior
EGF, progesterone and estrogen receptors.assay revealed significant reduction in cell viability
(Fig. 4), while MCF7 did not show due to mentioned supportive receptors in MCF7
(Rulli, Antognelli et al
STAT
MCF
STAT
BT20
ioImpacts
Quantitative real time PCR cells (Panel A) and BT20 cells
relative expressions intensity ratio of the genes upon the expression of housekeeping GAPDH
significant statistically SYBR Green emitted fluorescent
intensity in triplicate experiments statistically (mean ±
Although, various drugs have been proposed for the treatment of breast cancer, lack of deep knowledge about
adverse side effects of routine anticancer drugs are obstacles of complete Consequently, many efforts are in progress to find new approaches to overcome cancer.junctions is one of these efforts and CBX is a prototypic
However, in the case of CBX,RNA decreased in treated group in
of cells (data not shown) which could indicate of cell numbers
It should be highlighted that MCF7 cells have more proliferative and progressive behavior
EGF, progesterone and estrogen receptors.assay revealed significant reduction in cell viability
(Fig. 4), while MCF7 did not show due to mentioned supportive receptors in MCF7
et al
STAT5
MCF7
STAT5
20 Cells
ioImpacts, 2011, 1(2),
Quantitative real time PCR cells (Panel A) and BT20 cells
relative expressions intensity ratio of the genes upon the GAPDH
significant statisticallySYBR Green emitted fluorescent
intensity in triplicate experiments statistically (mean ±
Although, various drugs have been proposed for the lack of deep knowledge about
adverse side effects of routine anticancer drugs are obstacles of complete Consequently, many efforts are in progress to find new approaches to overcome cancer.junctions is one of these efforts and CBX is a prototypic
However, in the case of CBX,RNA decreased in treated group in
of cells (data not shown) which could indicate after incubation with drug
It should be highlighted that MCF7 cells have more proliferative and progressive behavior
EGF, progesterone and estrogen receptors.assay revealed significant reduction in cell viability
(Fig. 4), while MCF7 did not show due to mentioned supportive receptors in MCF7
et al. 2006)
CYCS
MCF7 Cells
CYCS
Cells
, 2011, 1(2),
Quantitative real time PCR assessments of CBX cells (Panel A) and BT20 cells (Panel B)
relative expressions intensity ratio of the genes upon the GAPDH gene.
significant statistically upon SYBR Green emitted fluorescent
intensity in triplicate experiments statistically (mean ±
Although, various drugs have been proposed for the lack of deep knowledge about
adverse side effects of routine anticancer drugs are obstacles of complete Consequently, many efforts are in progress to find new approaches to overcome cancer. junctions is one of these efforts and CBX is a prototypic
However, in the case of CBX,RNA decreased in treated group in
of cells (data not shown) which could indicate fter incubation with drug
It should be highlighted that MCF7 cells have more proliferative and progressive behavior
EGF, progesterone and estrogen receptors.assay revealed significant reduction in cell viability
(Fig. 4), while MCF7 did not show due to mentioned supportive receptors in MCF7
. 2006)
CYCS
Cells
CYCS
Cells
, 2011, 1(2),
assessments of CBX (Panel B)
relative expressions intensity ratio of the genes upon the gene.
upon GAPDH.SYBR Green emitted fluorescent
intensity in triplicate experiments statistically (mean ±
Although, various drugs have been proposed for the lack of deep knowledge about
adverse side effects of routine anticancer drugs are obstacles of complete Consequently, many efforts are in progress to find new
Inhibitionjunctions is one of these efforts and CBX is a prototypic
However, in the case of CBX,RNA decreased in treated group in
of cells (data not shown) which could indicate fter incubation with drug
It should be highlighted that MCF7 cells have more proliferative and progressive behavior
EGF, progesterone and estrogen receptors.assay revealed significant reduction in cell viability
(Fig. 4), while MCF7 did not show due to mentioned supportive receptors in MCF7
. 2006). According to the
BCL
Cells
BCL
Cells
, 2011, 1(2), 113-
assessments of CBX (Panel B)
relative expressions intensity ratio of the genes upon the gene. All of the genes
GAPDH.SYBR Green emitted fluorescent
intensity in triplicate experiments statistically (mean ±
Although, various drugs have been proposed for the lack of deep knowledge about
adverse side effects of routine anticancer drugs are obstacles of complete Consequently, many efforts are in progress to find new
Inhibitionjunctions is one of these efforts and CBX is a prototypic
However, in the case of CBX,RNA decreased in treated group in
of cells (data not shown) which could indicate fter incubation with drug
It should be highlighted that MCF7 cells have more proliferative and progressive behavior, depend
EGF, progesterone and estrogen receptors.assay revealed significant reduction in cell viability
(Fig. 4), while MCF7 did not show due to mentioned supportive receptors in MCF7
According to the
BCL2
BCL2
-119
assessments of CBX (Panel B). Normalized
relative expressions intensity ratio of the genes upon the All of the genes
GAPDH. The values SYBR Green emitted fluorescent
intensity in triplicate experiments statistically (mean ± SEM)
Although, various drugs have been proposed for the lack of deep knowledge about
adverse side effects of routine anticancer drugs are obstacles of complete treatmentConsequently, many efforts are in progress to find new
Inhibitionjunctions is one of these efforts and CBX is a prototypic
However, in the case of CBX,RNA decreased in treated group in
of cells (data not shown) which could indicate fter incubation with drug
It should be highlighted that MCF7 cells have more depend
EGF, progesterone and estrogen receptors.assay revealed significant reduction in cell viability
(Fig. 4), while MCF7 did not show due to mentioned supportive receptors in MCF7
According to the
2
2
assessments of CBX . Normalized
relative expressions intensity ratio of the genes upon the All of the genes
The values SYBR Green emitted fluorescent
SEM)
Although, various drugs have been proposed for the lack of deep knowledge about
adverse side effects of routine treatment
Consequently, many efforts are in progress to find new Inhibition of
junctions is one of these efforts and CBX is a prototypic However, in the case of CBX,
RNA decreased in treated group in of cells (data not shown) which could indicate
fter incubation with drugIt should be highlighted that MCF7 cells have more
dependentEGF, progesterone and estrogen receptors.
assay revealed significant reduction in cell viability (Fig. 4), while MCF7 did not show
due to mentioned supportive receptors in MCF7 According to the
AKT
AKT
| 117
assessments of CBX . Normalized
relative expressions intensity ratio of the genes upon the All of the genes
The values SYBR Green emitted fluorescent
SEM).
Although, various drugs have been proposed for the lack of deep knowledge about
adverse side effects of routine treatment
Consequently, many efforts are in progress to find new of gap
junctions is one of these efforts and CBX is a prototypic However, in the case of CBX, total
RNA decreased in treated group in of cells (data not shown) which could indicate
fter incubation with drugIt should be highlighted that MCF7 cells have more
ent MTT
assay revealed significant reduction in cell viability (Fig. 4), while MCF7 did not show such
due to mentioned supportive receptors in MCF7 According to the
AKT1
AKT1
117
assessments of CBX on
. Normalized relative expressions intensity ratio of the genes upon the
All of the genes The values
SYBR Green emitted fluorescent
Although, various drugs have been proposed for the lack of deep knowledge about
adverse side effects of routine treatment.
Consequently, many efforts are in progress to find new gap
junctions is one of these efforts and CBX is a prototypic otal
RNA decreased in treated group in of cells (data not shown) which could indicate
fter incubation with drug. It should be highlighted that MCF7 cells have more
on MTT
assay revealed significant reduction in cell viability in such
due to mentioned supportive receptors in MCF7 According to the
Asadi-Khiavi et al.
118 | BioImpacts, 2011, 1(2), 113-119 Copyright © 2011 by Tabriz University of Medical Sciences
results, CBX inhibits MCF7 and BT20 cells growth and viability just in a dose dependent manner with IC50 values of 150 μM after 24 h of exposure. The anti-ploriferative effects of CBX have been reported in other cancer cells (Kawashima et al. 2009). Apoptotic effects of CBX have been also reported in some types of cells (Pivato et al. 2006; Waddell et al. 2000). Among several mechanisms of action which have been proposed for pharmacological activity of CBX, induction of Mitochondrial Permeability Transition (MPT) and release of cytochrome c is prominent (Salvi et al. 2005; Pivato et al. 2006). Therefore, it can be considered as an apoptosis stimulating drug (Isbrucker et al. 2006; Salvi et al. 2005). Due to structural similarity to glucocorticoids, CBX inhibits Hydroxysteroid dehydrogenases (HSD) in cancer cells and induces apoptosis via interaction with glucocorticoid and mineralocorticoid receptors (Greenstein et al. 2002; Voutsas et al. 2007). It seems that TP53 and CYCS genes as well as STAT1 gene down regulation are compensatory responses against anti-apoptotic genes down regulation which totally arise from stress oxidative effect of Carbenoxolone (Farczadi, Kaszas et al. 2002). AKT1 and BCL2 genes are anti-apoptotic down regulated genes with similar effect on cancer cells. Of these down regulated genes, the BCL2 gene encodes the bcl2 protein that involved in a wide variety of cellular activities such as homeostasis and tumorigenesis. The encoded protein is able to reduce the release of pro-apoptotic cytochrome c from mitochondria and block caspase activation. This suggests a cytoprotective and cell survival functionality for this gene as reported previously ( Karsan et al. 1996; Choi et al. 1995) but as it mentioned, cytochrome c has been down regulated in CBX group, probably due to carbenoxolone stress oxidative effect (Salvi et al. 2005; Pivato et al. 2006) and BCL2 counter regulatory gene, was not able to be compensated. In the view of transcryptomics, it seems that Carbenoxolone affects cancer cell viability despite some ambiguous findings in cell viability and cytotoxicity assays. Lewenstein and his co-workers provided the “Homologous Growth Control (HGC)” theory approximately forty years ago which talks about gap junctions' delirious role in carcinogenesis particularly in the case of gap junction intercellular communication(GJIC) between normal and cancer cells (Cronier, Crespin et al. 2009) and CBX has confirmed inhibitory effects on gap junction-mediated intercellular communication (GJIC) particularly in attached cells as well (Winmill et al. 2003; Paraguassu-Braga et al. 2003). Therefore we cannot establish a precise mechanism for CBX activity.
Conclusion
Breast cancer has emerged as a leading cause of cancer death in the world particularly in women. Unfortunately,
the current therapeutic strategies are ineffective in some cases; hence, there exists an increasing demand for more effective therapies. We examined the CBX in this matter. In conclusion, based on the results obtained in this study, we suggest that CBX be favourable for in vivo applications; nevertheless further investigations are yet to be fulfilled to authenticate its in vivo usefulness. This obviously means that there is a novel relationship between cell-cell communications and inter-cellular cross talking, perhaps via gap junctions, at whom we propose that interestingly orchestrated biochemical machinery of cells may be related to some downstream cell signalling which may play a key role in development of cancer. On the other hand, the main question is remained: Are gap junctions friend or enemy? Therefore we propose evaluation of CBX effects on genetically modified cancer cell lines particularly in transcriptomics and functional genomics level using DNA chip technology.
Ethical issues None to be declared. Conflict of interests The authors declare no conflict of interests. Acknowledgement Authors would like to thank Research Center for Pharmaceutical Nanotechnology (RCPN), Tabriz University of Medical Sciences, Tabriz, Iran for the financial support of this project (grant No. 87011) that is a part of PhD thesis number 32 Also authors are thankful to Dr Y. Omidi for his kind support.
References Afaq F, Zaman N, Khan N, Syed D. N, Sarfaraz S, Zaid M. A and Mukhtar H. 2008. Inhibition of epidermal growth factor receptor signaling pathway by delphinidin, an anthocyanidin in pigmented fruits and vegetables. Int. J. Cancer. 123, 1508
Agelaki S, Spiliotaki M, Markomanolaki H, Kallergi G, Mavroudis D, Georgoulias V and Stournaras C. 2009. Caveolin-1 regulates EGFR signalling in MCF-7 breast cancer cells and enhances gefitinib-induced tumor cell inhibition. Cancer Biol. Ther, 8(15), 1470-1477.
Cameron SJ, Malik S, Akaike M, Lerner-Marmarosh N, Yan C, Lee JD, Abe J, Yang J. 2003. Regulation of epidermal growth factor-induced connexin 43 gap junction communication by big mitogen-activated protein kinase1/ERK5 but not ERK1/2 kinase activation. J Biol Chem, 278(20), 18682-18688.
Camp E. R, Summy J, Bauer T. W, Liu W, Gallick G. E and Ellis L. M. 2005. Molecular mechanisms of resistance to therapies targeting the epidermal growth factor receptor. Clin. Cancer Res. 11, 397.
Gap junctions in cancer cells
Copyright © 2011 by Tabriz University of Medical Sciences BioImpacts, 2011, 1(2), 113-119 | 119
Cronier, L., S. Crespin, et al. 2009. Gap junctions and cancer: new functions for an old story. Antioxid Redox Signal. 11(2): 323-338.
Dong J and Wiley HS. 2000. Trafficking and proteolytic release of epidermal growth factor receptor ligands are modulated by their membrane-anchoring domains. J Biol Chem. 275(1), 557-64.
Farczadi, E., I. Kaszas, et al. 2002. Changes in apoptosis, mitosis, Her-2, p53 and Bcl2 expression in breast carcinomas after short-term tamoxifen treatment. Neoplasma 49(2), 101-103.
Gialeli C, Kletsas D, Mavroudis D, Kalofonos H. P, Tzanakakis G. N and Karamanos N. K. 2009. Targeting epidermal growth factor receptor in solid tumors: Critical evaluation of the biological importance of therapeutic monoclonal antibodies. Curr. Med. Chem. 16, 3797.
Greenstein S, Ghias K, Krett NL. and Rosen ST. 2002. Mechanisms of glucocorticoid-mediated apoptosis in hematological malignancies. Clin Cancer Res. 8, 1681-94.
Isbrucker RA, Burdock GA. 2006. Risk and safety assessment on the consumption of Licorice root (Glycyrrhiza sp), its extract and powder as a food ingredient, with emphasis on the pharmacology and toxicology of glycyrrhizin. Regul Toxicol Pharmacol. 46, 167–92.
Kawashima D, Asai M, Katagiri K, Takeuchi R, Ohtsuka K. 2009. Reinvestigation of the effect of carbenoxolone on the induction of heat shock proteins. Cell Stress and Chaperones. 14: 535-43.
Leithe E, Sirnes S, Omori Y, Rivedal E. 2006. Downregulation of gap junctions in cancer cells. Crit Rev Oncog, 12(4), 225-256.
Li, J., et al. 2001. Upregulation of gap junction connexin 32 with epileptiform activity in the isolated mouse hippocampus. Neuroscience. 105(3), 589-598.
Loew S, Schmidt U, Unterberg A and Halatsch M. E. 2009. The epidermal growth factor receptor as a therapeutic target in glioblastoma multiforme and other malignant neoplasms. Anticancer Agents Med. Chem. 9, 703.
Mamot C and Rochlitz C. 2006. Targeting the epidermal growth factor receptor (EGFR)–a new therapeutic option in oncology. Swiss. Med. Wkly. 136, 4.
May, P. and May, E. 1999. Twenty years of p53 research: structural and functional aspects of the p53 protein. Oncogene. 18, 7621–36.
Nicholson BJ. 2003. Gap junctions: from cell to molecule. J. Cell Sci. 116(22), 4479-81.
Paraguassu-Braga FH, Borojevic R, Bouzas LF, Barcinski MA, Bonomo A. 2003. Bon marrow stroma inhibits proliferation and apoptosis in leukemic cells through gap junction-mediated cell communication, Cell Death Differ. 10: 1101–8.
Pfaffl MW. 2001.A new mathematical model for relative quantification in real-time RT-PCR. Nucleic Acids Res. 29(9), 45.
Pivato LS, Constantin RP, Ishii-Iwamoto EL, Yamamoto NS, Constantin J, Bracht A. 2006. Metabolic effects of carbenoxolone in the rat liver. J Biochem Mol Toxicol. 20:230-40.
Rulli, A., C. Antognelli, et al. 2006. A possible regulatory role of 17beta-estradiol and tamoxifen on glyoxalase I and glyoxalase II genes expression in MCF7 and BT20 human breast cancer cells. Breast Cancer Res Treat. 96(2): 187-196.
Salvi M, Fiore C, Battaglia V, Palermo M, Armanini D, Toninello A. 2005. Carbenoxolone induces oxidative stress in Liver mitochondria, which is responsible for transition pore opening. Endocrinol. 146, 2306-12.
Voutsas IF, Gritzapis AD, Alexis MN, Katsanou ES, Perez S, Baxevanis CN, Papamichail M. 2007. A novel quantitative flow cytometric method for measuring glucocorticoid receptor (GR) in cell lines: Correlation with the biochemical determination of GR. J Immunol Methods. 324: 110-19.
Waddell BJ, Susan Hisheh S, Dharmarajan AM, Burton PJ. 2000. Apoptosis in rat placenta is zone-dependent and stimulated by glucocorticoids. BOR. 63: 1913-17.
Winmill RE, Hedrick MS. 2003. Gap junction blockade with Carbenoxolone differentially affects fictive breathing in larval and adult bullfrogs. Respir Physiol Neurobiol. 138: 239-/51.
Ye, Z. C., et al. 2003. Functional hemichannels in astrocytes: a novel mechanism of glutamate release. J. Neurosci. 23(9), 3588-3596.